Attenuated FNR deficient enterobacteria

ABSTRACT

The invention provides an attenuated enterobacterium comprising an attenuating mutation in the fnr gene, and optionally further comprising a heterologous nucleic acid encoding a foreign antigen. Also provided are pharmaceutical formulations comprising the attenuated enterobacteria of the invention. Further disclosed are methods of inducing an immune response in a subject by administration of an immunogenically effective amount of an attenuated enterobacterium or pharmaceutical formulation of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/500,366filed Jul. 9, 2009 (now U.S. Pat. No. 8,101,168), which is a divisionalof U.S. application Ser. No. 11/780,358 filed Jul. 19, 2007 (nowabandoned), which claims the benefit of U.S. Provisional Application No.60/831,821, filed Jul. 19, 2006, the disclosures of which areincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates to attenuated Fumarate-Nitrate Reductase (FNR)enterobacteria strains. In particular, this invention relates toattenuated FNR enterobacteria strains and methods of using the same toinduce an immune response.

BACKGROUND OF THE INVENTION

Salmonella enterica serovar Typhimurium is a gram-negative facultativeintracellular pathogen. Serovar Typhimurium infections usually resultfrom ingestion of contaminated food or water. The organism generallytargets and colonizes the intestinal epithelium of the host and causesgastroenteritis (i.e., salmonellosis). During a Salmonella infection,the growth phase and growth conditions of the organism are important inattachment, invasion, and the regulation of many of the virulence genes.Cells grown under limited oxygen concentrations are more invasive andadhere better to mammalian cells than do aerobically grown orstationary-phase cells. Salmonella invasion genes have been identifiedand localized. During infection, serovar Typhimurium must adapt tochanges in [O₂] encountered in the gastrointestinal tract of the host.In Escherichia coli, transitions from aerobic to anaerobic environmentsor vice versa, involve changes in a large number of genes. However, uponsudden reappearance of oxygen, these cellular processes must be reversedin a precise and orderly fashion to ensure the safe transition to theoxygenated environment. This complex regulatory system has beenextensively studied in E. coli, where the DNA-binding protein FNRencoded by fnr, senses changes in [O₂] and controls the expression ofthe different genes either alone or in cooperation with otherregulators, e.g., ArcA.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the inventors' discoverythat enterobacteria comprising an attenuating mutation in the fnr(Fumarate-Nitrate Reductase) gene have an avirulent (i.e., highlyattenuated) phenotype. Thus, the present invention provides attenuatedenterobacteria and methods of using the same as attenuated immunogeniccompositions, attenuated vaccines and/or as attenuated vaccine vectorsto induce an immune response against a heterologous antigen in asubject.

Accordingly, a first aspect of the invention provides a pharmaceuticalcomposition comprising an attenuated enterobacterium comprising anattenuating mutation (e.g., deletion) in the fnr gene in apharmaceutically acceptable carrier.

A further aspect of the invention provides an attenuated enterobacteriumcomprising an attenuating mutation (e.g., a deletion) in the fnr geneand, optionally, further comprising a heterologous nucleic acid sequenceencoding a foreign antigen. In particular embodiments, the attenuatedenterobacterium is present in a pharmaceutical composition in apharmaceutically acceptable carrier.

A further aspect of the present invention is a method of inducing animmune response in a subject comprising administering to the subject animmunogenically effective amount of an attenuated enterobacteriumcomprising an attenuating mutation (e.g., a deletion) in the fnr gene.In embodiments of the invention, the attenuated enterobacterium isprovided in a pharmaceutical composition further comprising apharmaceutically acceptable carrier. In further embodiments of theinvention, the attenuated enterobacterium comprises a heterologousnucleic acid encoding a foreign antigen.

The invention further provides for the use of an attenuatedenterobacterium or pharmaceutical composition of the invention to inducean immune response in a subject.

These and other aspects of the invention are set forth in more detail inthe following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the location of the tnpA insertion (between bp 106 and 107)in the fnr gene. WT fnr sequences are in bold, and the sequences of thebeginning and ending junctions of the tnpA insert are in italics. Arrowsindicate the direction of transcription. IGS, intergenic spacer region.(Complete DNA sequences [i.e., ogt, tnpA/fnr junctions, and ydaA] areavailable at GenBank accession number AH015911.)

FIG. 2 shows a logo graph of the information matrix obtained from theconsensus alignment of FNR motif sequences for serovar Typhimurium(derived from the corresponding FNR-regulated genes in E. coli). Thetotal height of each column of characters represents the amount ofinformation for that specific position, and the height of each characterrepresents the frequency of each nucleotide.

FIG. 3 shows the correlation between the microarray and qRT-PCR data for19 selected genes. The ratios of changes in gene expression, from themicroarray and qRT-PCR experiments, for the FNR mutant relative to theWT were log₂ transformed and linearly correlated.

FIG. 4 shows a scheme representing the structural organization of themajor genes involved in virulence/SPI-1 (A), ethanolamine utilization(B), and flagellar biosynthesis and motility/swarming (C to E). Thenames of genes are listed to the right of the arrows, an asterisk nextto the gene indicates the presence of at least one FNR motif in the 5′region, and the numbers to the left of the arrows indicate the ratio ofgene expression in the fnr mutant relative to that in the WT.

FIG. 5 shows a comparison of the fnr mutant and the WT strain forvirulence in 6- to 8-week-old C57BL/6 mice. (A) Groups of 10 mice wereinoculated p.o. with 5×10⁶ and 5×10⁷ CFU/mouse. (B) Groups of five micewere challenged i.p. with 250 CFU/mouse, as described. Percent survivalis the number of mice surviving relative to the number of micechallenged at time zero.

FIG. 6 shows a comparison of the WT, the fnr mutant, and the mutantstrain harboring pfnr for survival inside peritoneal macrophages fromC57BL/6 mice. The macrophages were harvested and treated as described.(A) Comparison between the fnr mutant and the WT strain. The number ofviable cells found inside the macrophages, at time zero, following theremoval of extracellular bacteria by washing/gentamicin treatment isdefined as 100% survival. (B) Comparison between the WT, the fnr mutant,and the pfnr-complemented mutant. The number of viable cells foundinside macrophages at 20 h is expressed as percent survival relative tothat found inside macrophages at 2 h.

FIG. 7 shows the virulence of the WT and the fnr mutant in C57BL/6 miceand congenic gp91phox^(−/−) mice and survival of the bacteria insideperitoneal macrophages. The mice were challenged i.p. with 250CFU/mouse, as described. (A) C57BL/6 and gp91phox^(−/−) mice treatedwith the WT strain. (B) C57BL/6 and gp91phox^(−/−) mice treated with thefnr mutant. (C) Survival of the WT and the fnr mutant inside macrophagesfrom C57BL/6 and gp91phox^(−/−) mice. The number of viable cells at 20 his expressed as percent survival relative to that found inside themacrophages at time zero.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure, which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations, and variations thereof.

All publications, patents, and patent publications cited herein areincorporated by reference in their entireties for the teachings relevantto the sentence and/or paragraph in which the citation is presented.

As used herein, “a,” “an” or “the” can mean one or more than one. Forexample, “a” mutation can mean a single mutation or a multiplicity ofmutations.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

FNR (Fumarate-Nitrate Reductase) is a DNA-binding regulator proteinexpressed by all enterobacteria including Salmonella spp., Escherichiaspp., and Shigella spp. The fnr gene was previously known as oxrA inSalmonella spp. The present inventors have identified FNR as a globalregulatory protein for the expression of virulent genes inenterobacteria. An FNR deleted (Δfnr) strain of a known virulent strainof S. enterica serovar Typhimurium was shown to be non-motile, lackingflagella, and having an avirulent phenotype. Thus, the present inventionprovides FNR deficient (e.g., Δfnr or fnr mutants) strains ofenterobacteria that can be used to study the fnr gene, its role invirulence in these organisms, and can further be used as attenuatedimmunogenic compositions, attenuated vaccines (e.g., live attenuatedvaccines) and/or attenuated vaccine vectors.

Enterobacteria are known in the art and are generally pathogens that caninfect the gastrointestinal tract of avians and/or mammals. The presentinvention can be practiced with any suitable enterobacterium in theorder Enterobacteriales and optionally in the family Enterobacteriaceaethat encodes FNR, including but not limited to bacteria classified inthe following genera: Alishewanella, Alterococcus, Aquamonas, Aranicola,Arsenophonus, Azotivirga, Blochmannia, Brenneria, Buchnera, Budvicia,Buttiauxella, Candidatus, Cedecea, Citrobacter, Dickeya, Edwardsiella,Enterobacter, Erwinia (e.g., E. amylovora), Escherichia, Ewingella,Grimontella, Hafnia, Klebsiella (e.g., K. pneumoniae), Kuyvera,Leclercia, Leminorella, Moellerella, Morganella, Obesumbacterium,Pantoea, Pectobacterium, Candidatus Phlomobacter, Photohabdus,Plesiomonas (e.g., P. shigelloides), Pragia, Proteus (e.g., P.vulgaris), Providencia, Rahnella, Raoultella, Salmonella, Samsonia,Serratia (e.g., S. marcenscens), Shigella, Sodalis, Tatumella,Travulsiella, Wigglesworthia, Xenorhabdus, Yersinia (e.g., Y. pestis),and Yokenella.

In particular embodiments, the enterobacterium is a Salmonella spp., anEscherichia spp., or a Shigella spp.

Further, the enterobacterium can optionally be a pathogenicenterobacterium. In particular embodiments, the enterobacterium fromwhich the FNR deficient strain is derived is a pathogenic (e.g.,virulent) bacterium as that term is understood in the art, where theattenuating fnr mutation results in a reduction in the pathogenicity. Inrepresentative embodiments, the FNR deficient strain is highlyattenuated so as to be avirulent (e.g., induces no or insignificantlevels of pathogenicity).

The term “pathogenic” is understood in the art, for example, as causingpathogenicity such as morbidity and/or mortality in a subject orpopulation of subjects.

The term “attenuating” with respect to pathogenic microorganisms isunderstood in the art, for example, as a reduction in pathogenicity(including no detectable pathogenicity) produced in the subject as aresult of administration of the FNR deficient enterobacterium strain ascompared with the level of pathogenicity produced if an enterobacteriumwith a fully functional fnr gene (e.g., the wild-type strain) wereadministered.

Methods of assessing pathogenicity of enterobacteria, and attenuationthereof, are known in the art (e.g., morbidity and/or mortalityfollowing challenge in a suitable animal model such as mice or survivalin cultured macrophages).

Suitable Salmonella species within the scope of the present inventioninclude but are not limited to S. bongori and S. enterica as well as S.enterica subspecies (e.g., enterica, salamae, arizonae, diarizonae,houtenae and indica). Numerous serovars of S. bongori and S. entericaare known and are within the scope of the present invention. ExemplaryS. enterica serovars include Typhimurium, Typhi and Enteritidis.

The present invention can further be practiced with any species ofEscherichia including but not limited to E. adecarboxylata, E. albertii,E. blattae, E. coli (including toxigenic strains such as E. coliO157:H7), E. fergusonii, E. hermannii, and E. vulneris.

Suitable species of Shigella include without limitation species inSerogroup A (e.g., S. dysenteriae and serotypes thereof), species inSerogroup B (e.g., S. flexneri and serotypes thereof), species inSerogroup C (e.g., S. boydii and serotypes thereof), and species inSerogroup D (e.g., S. sonnei and serotypes thereof).

The genomic sequences of numerous enterobacteria are known in the art.See, e.g., NCBI Accession No. NC_(—)004337 (Shigella flexneri 2a str.301); NCBI Accession No. NC_(—)007613 (Shigella boydii Sb227); NCBIAccession No. AP009048 (E. coli W3110); NCBI Accession No. BA000007 (E.coli O157:H7 str. Sakai); NCBI Accession No. AE009952 (Y. pestis KIM);NCBI Accession No. NC_(—)003197 (S. typhimurium LT2); and NCBI AccessionNo. NC_(—)003198 (S. enterica subsp. enterica serovar Typhi str. CT18).

Likewise, the nucleic acid and amino acid sequences of the fnr gene fromvarious enterobacteria are known in the art.

The attenuated enterobacteria of the present invention comprise anattenuating mutation in the fnr gene. In representative embodiments, themutation is an attenuating deletion mutation (including truncations)that results in attenuation of the pathogenicity of the bacterium. Othermutations include without limitation attenuating insertions,substitutions and/or frame-shift mutations that result in attenuation ofthe pathogenicity of the bacterium. In embodiments of the invention, themutation is a non-polar alteration in the fnr gene.

Deletion and insertion mutations can be any deletion/insertion mutationin the fnr gene that results in attenuation of the pathogenicity of thebacterium. In representative embodiments, the alteration is a deletionor an insertion of at least about 9, 30, 50, 75, 90, 120, 150, 180, 240,300, 450 or more consecutive nucleotides in the fnr gene that results inattenuation of the pathogenicity of the bacterium. Optionally,essentially all (e.g., at least about 95%, 97%, 98% or more) or all ofthe fnr coding sequence is deleted. In other embodiments, essentiallyall or all of the fnr gene, including regulatory elements, is deleted.In particular embodiments, the deletion can extend beyond the fnr gene.Generally, however, the deletion does not render genes essential forgrowth, multiplication and/or survival non-functional. In particularembodiments, the deletion does not extend into any genes essential forgrowth, multiplication and/or survival. In embodiments of the invention,the deletion does not extend to genes that are 5′ and/or 3′ of the fnrcoding region or the fnr gene.

One FNR deficient strain of S. enterica serovar Typhimurium has beenconstructed by the inventors and is shown in the Examples.

The FNR deficient enterobacterium strains of the invention can furthercomprise other mutations, including other attenuating mutations.

Generally, the FNR deficient enterobacterium strains will retain otherappropriate genomic sequences to be able to grow, multiply and survive(e.g., in the gut of a host). Thus, the fnr mutations of the inventionexclude lethal mutations that unduly inhibit the survival of theorganism.

In embodiments of the invention, the FNR mutation results in at leastabout a 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or morereduction in FNR mRNA, protein and/or activity. Methods of assessinglevels of mRNA and proteins levels and FNR activity are known in theart.

The fnr mutation can be combined with any other mutation known in theart, including other attenuating mutations. For example, the fnr mutantenterobacterium can also comprise an arcA mutation. The ArcA proteincooperates with FNR for controlling the transitions fromaerobic/anaerobic conditions and vice versa.

The attenuated enterobacteria of the present invention can be used as anattenuated immunogenic compositions or attenuated vaccine againstenterobacteria (e.g., a live attenuated vaccine). Enterobacteria aredescribed above. In embodiments of the invention, the enterobacterium isa pathogenic enterobacterium. For example, in particular embodiments,the invention provides live immunogenic compositions or live attenuatedvaccines against Salmonella, Shigella or Escherichia. The attenuatedenterobacterium vaccine can be used to induce an immune response againstone species or against multiple closely related species/genera ofenterobacteria (e.g., that cross-react with antibodies produced inresponse to administration of the attenuated enterobacterium).

Further, the attenuated FNR deficient enterobacterium strains of theinvention can be used as vectors, e.g., to deliver an antigen(s) that isheterologous (e.g., foreign) to the enterobacterium vector (includingany plasmids carried by the enterobacterium) to induce an immuneresponse against other organisms (e.g., pathogenic organisms). In oneembodiment, a heterologous nucleic acid sequence encoding the foreignantigen(s) is incorporated into the genomic DNA of the enterobacterium(e.g., inserted into or in place of a deleted fnr gene). In otherembodiments, the heterologous nucleic acid sequence encoding the foreignantigen is incorporated into a plasmid that is carried by an attenuatedFNR deficient host (e.g., a Δfnr host). Plasmids that are compatiblewith the various enterobacteria are known in the art.

The attenuated FNR deficient strains can further be used as vectors todeliver therapeutic proteins and untranslated RNAs (e.g., siRNA, shRNA,antisense RNA).

Methods of expressing foreign antigens in enterobacteria are known tothose skilled in the art. For example, the foreign antigen can beexpressed as part of a fusion with one of the structural proteins of thebacterial host (e.g., expressed on the surface of the bacterium) such asa flagellin protein (see, e.g., Chauhan et al., (2005) Molecular andCellular Biochemistry 276:1-6) or a membrane protein as known in theart. See also, Chinchilla et al., (2007) Infection Immun. 75: 3769. Inother embodiments, the foreign antigen is not expressed as a fusion witha host structural protein. According to this embodiment, theheterologous nucleic acid encoding the foreign antigen can optionally beoperably associated with a leader sequence directing secretion, of theforeign antigen from the bacterial cell.

The heterologous nucleic acid sequence encoding the foreign antigen canbe operatively associated with any suitable promoter or other regulatorysequence. The promoter or regulatory sequence can be native or foreignto the host, can be native or foreign to the heterologous nucleic acid,and can further be partially or completely synthetic.

The codon usage of the heterologous nucleic acid sequence can beoptimized for expression in the enterobacterium using methods known tothose skilled in the art (see, e.g., Chinchilla et al., (2007) InfectionImmun. 75: 3769.

The foreign antigen can be any suitable antigen known in the art, andcan further be from a bacterial, yeast, fungal, protozoan or viralsource. Suitable antigens include, but are not limited to antigens frompathogenic infectious agents.

The antigen can be an antigen from a pathogenic microorganism, whichincludes but is not limited to, Rickettsia, Chlamydia, Mycobacteria,Clostridia, Corynebacteria, Mycoplasma, Ureaplasma, Legionella,Shigella, Salmonella, pathogenic Escherichia coli species, Bordatella,Neisseria, Treponema, Bacillus, Haemophilus, Moraxella, Vibrio,Staphylococcus spp., Streptococcus spp., Campylobacter spp., Borreliaspp., Leptospira spp., Erlichia spp., Klebsiella spp., Pseudomonas spp.,Helicobacter spp., and any other pathogenic microorganism now known orlater identified (see, e.g., Microbiology, Davis et al, Eds., 4^(th)ed., Lippincott, N.Y., 1990, the entire contents of which areincorporated herein by reference for the teachings of pathogenicmicroorganisms).

Specific examples of microorganisms from which the antigen can beobtained include, but are not limited to, Helicobacter pylori, Chlamydiapneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasmapneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Streptococcuspneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseriameningitidis, Neisseria gonorrhoeae, Treponema pallidum, Bacillusanthracis, Salmonella typhi, Vibrio cholera, Pasteurella pestis,Pseudomonas aeruginosa, Campylobacter jejuni, Clostridium difficile,Clostridium tetani, Clostridium botulinum, Mycobacterium tuberculosis,Borrelia burgdorferi, Haemophilus ducreyi, Corynebacterium diphtheria,Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenza, and enterotoxic Escherichia coli.

The antigen can further be an antigen from a pathogenic protozoa,including, but not limited to, Plasmodium spp. (e.g., malaria antigens),Babeosis spp., Schistosoma spp., Trypanosoma spp., Pneumocystis carnii,Toxoplasma spp., Leishmania spp., and any other protozoan pathogen nowknown or later identified.

The antigen can also be an antigen from pathogenic yeast and fungi,including, but not limited to, Aspergillus spp., Candida spp.,Cryptococcus spp., Histoplasma spp., Coccidioides spp., and any otherpathogenic fungus now known or later identified.

Suitable antigens can include, but are not limited to, viral antigenssuch as antigens including but not limited to human hepatitis C virus(HCV) antigens and influenza antigens.

Other specific examples of various antigens include, but are not limitedto, the B1 protein of hepatitis C virus (Bruna-Romero et al. (1997)Hepatology 25: 470-477), amino acids 252-260 of the circumsporozoiteprotein of Plasmodium berghei [Allsopp et al. (1996) Eur. J. Immunol.26: 1951-1958], the influenza A virus nucleoprotein [e.g., residues366-374; Nomura et al. (1996) J. Immunol. Methods 193: 4149], thelisteriolysin O protein of Listeria monocytogenes [residues 91-99; An etal. (1996) Infect. Immun. 64: 1685-1693], P. falciparum antigens(causing malaria, e.g., tCSP), hepatitis B surface antigen [Gilbert etal. (1997) Nature Biotech. 15: 1280-1283], and E coli O157.H1.

The term “antigen” as used herein includes toxins such as the neurotoxintetanospasmin produced by Clostridium tetani and the toxin produced byE. coli O157:H7.

In particular embodiments, the attenuated enterobacteria of theinvention express a foreign antigen(s) and can be used to induce animmune response against both the enterobacterium and the organism(s)from which the foreign antigen(s) is derived and, optionally, otherspecies/genera closely related to either of the foregoing (e.g., thatcross-react with antibodies produced in response to administration ofthe attenuated enterobacterium).

There is no particular size limitation to the heterologous nucleic acidencoding the foreign antigen. When incorporated into the genomic DNA,the heterologous nucleic acid will generally be at least about 30, 50,75, 100, 150 or 200 nucleotides in length and/or less than about 1, 1.5,2, 2.5 or 3 kilobases in length. When carried by a plasmid, theheterologous nucleic acid can generally be longer, e.g., at least about30, 50, 75, 100, 150, 200, 500 or 1000 nucleotides in length and/or lessthan about 5, 10, 12, 14, 16, 18 or 20 kilobases in length.

In representative embodiments, the FNR deficient enterobacterium is aΔfnr mutant, which advantageously reduces the probability of reversionto the wild-type pathogenic phenotype. For example, most current liveattenuated vaccine strains against typhoid are auxotrophs for somenutrients, which are likely less stable than the deletion mutants.

The present invention can be used for therapeutic/prophylactic andnon-therapeutic/prophylactic purposes. For example, the presentinvention provides FNR deficient (e.g., Δfnr) enterobacteria strainsthat can be used to study the fnr gene, its role in virulence in theseorganisms, and as attenuated immunogenic compositions, attenuatedvaccines (e.g., live attenuated vaccines) and attenuated vaccine vectors(e.g., live attenuated vaccine vectors).

With respect to uses as an attenuated vaccine or vaccine vector, thepresent invention finds use in both veterinary and medical applications.Suitable subjects include avians, mammals and fish, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys and pheasants. The term“mammal” as used herein includes, but is not limited to, primates (e.g.,simians and humans), bovines, ovines, caprines, porcines, equines,felines, canines, lagomorphs, rodents (e.g., rats and mice), etc. Humansubjects include fetal, neonatal, infant, juvenile and adult subjects.

The invention can be used in a therapeutic and/or prophylactic manner.For example, in one embodiment, to protect against an infectiousdisease, subjects may be vaccinated prior to exposure, e.g., as neonatesor adolescents. Adults that have not previously been exposed to thedisease may also be vaccinated.

In particular embodiments, the present invention provides apharmaceutical composition comprising a FNR deficient (e.g., Δfnr)strain enterobacterium (optionally, a live FNR deficiententerobacterium) in a pharmaceutically-acceptable carrier, which canalso include other medicinal agents, pharmaceutical agents, carriers,adjuvants, diluents, etc. For injection, the carrier is typically aliquid. For other methods of administration, the carrier may be eithersolid or liquid, such as sterile, pyrogen-free water or sterilepyrogen-free phosphate-buffered saline solution. For inhalationadministration, the carrier will be respirable, and is optionally insolid or liquid particulate form. Formulation of pharmaceuticalcompositions is well known in the pharmaceutical arts [see, e.g.,Remington's Pharmaceutical Sciences, 15th Edition, Mack PublishingCompany, Easton, Pa. (1975)].

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, e.g., the material may beadministered to a subject without causing undesirable biologicaleffects.

The FNR deficient strains of the invention can be administered to elicitan immune response. Typically, immunological compositions of the presentinvention comprise an immunogenically effective amount of the FNRdeficient strain enterobacterium as disclosed herein, optionally incombination with a pharmaceutically acceptable carrier.

An “immunogenically effective amount” is an amount that is sufficient toinduce an immune response in the subject to which the composition isadministered. Nonlimiting examples of dosages include about 10⁴ to 10⁹colony forming units (cfu), about 10⁵ to 10⁸ cfu or about 10⁶ to 10⁷cfu. Optionally, one or more booster dosages (e.g., about 10³ to 10⁸ cfuor 10⁴ to 10⁵ cfu) can be administered.

The invention also encompasses methods of producing an immune responsein a subject, the method comprising: administering a FNR deficient(e.g., Δfnr) enterobacterium strain of the invention or a pharmaceuticalformulation containing the same to a subject in an immunogenicallyeffective amount so that an immune response is produced in the subject.

The terms “vaccination” or “immunization” are well-understood in theart. For example, the terms vaccination or immunization can beunderstood to be a process that increases a subject's immune reaction toantigen and thereby enhance the ability to resist and/or overcomeinfection.

Any suitable method of producing an immune response (e.g., immunization)known in the art can be employed in carrying out the present invention,as long as an active immune response (preferably, a protective immuneresponse) is elicited.

In representative embodiments, less pathogenicity (including nodetectable pathogenicity) is produced in the subject as a result ofadministration of the FNR deficient enterobacterium strain as comparedwith pathogenicity produced if an enterobacterium with a fullyfunctional fnr gene (e.g., the wild-type strain) were administered(e.g., at least about a 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%,97%, 98% or more reduction in pathogenicity).

Vaccines can be given as a single dose schedule or in a multiple doseschedule. A multiple dose schedule is one in which a primary course ofadministration may consist of about 1 to 10 separate doses, followed byother doses (i.e., booster doses) given at subsequent time intervals tomaintain and/or reinforce the immune response, for example, at about 1to 4 months for a second dose, and if needed, a subsequent dose(s) afteranother several months or year. The dosage regimen will also, at leastin part, be determined by the need of the individual and be dependentupon the judgment of the medical or veterinary practitioner.

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure toimmunogen by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease, the progressionof the disease and/or the symptoms of the disease. Alternatively, aprotective immune response or protective immunity may be useful in thetreatment of disease including infectious disease. The protectiveeffects may be complete or partial, as long as the benefits of thetreatment outweigh any disadvantages thereof.

Administration of the attenuated enterobacteria and compositions of theinvention can be by any means known in the art, including oral, rectal,topical, buccal (e.g., sub-lingual), vaginal, intra-ocular, parenteral(e.g., subcutaneous, intramuscular including skeletal muscle, cardiacmuscle, diaphragm muscle and smooth muscle, intradermal, intravenous,intraperitoneal), topical (e.g., mucosal surfaces including airwaysurfaces), intranasal, transmucosal, intratracheal, transdermal,intraventricular, intraarticular, intrathecal and inhalationadministration.

The most suitable route in any given case will depend on the nature andseverity of the condition being treated, the FNR deficient strainenterobacterium, and the composition being administered.

The FNR deficient enterobacterium strain can be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science and Practice of Pharmacy(9^(th) Ed. 1995). In the manufacture of a pharmaceutical formulationaccording to the invention, the FNR deficient enterobacterium strain istypically admixed with, inter alia, an acceptable carrier. The carriercan be a solid or a liquid, or both, and is optionally formulated as aunit-dose formulation, which can be prepared by any of the well-knowntechniques of pharmacy.

For injection, the carrier is typically a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). Forother methods of administration, the carrier can be either solid orliquid.

For oral administration, the FNR deficient enterobacterium strain can beadministered in solid dosage forms, such as capsules, tablets, andpowders, or in liquid dosage forms, such as elixirs, syrups, andsuspensions. The FNR deficient strain enterobacterium can beencapsulated in gelatin capsules together with inactive ingredients andpowdered carriers, such as glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate and the like. Examples ofadditional inactive ingredients that can be added to provide desirablecolor, taste, stability, buffering capacity, dispersion or other knowndesirable features are red iron oxide, silica gel, sodium laurylsulfate, titanium dioxide, edible white ink and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar coated or film coated to mask any unpleasant tasteand protect the tablet from the atmosphere, or enteric-coated forselective disintegration in the gastrointestinal tract. Liquid dosageforms for oral administration can contain coloring and flavoring toincrease patient acceptance.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the FNR deficient enterobacterium strain in aflavored base, usually sucrose and acacia or tragacanth; and pastillescomprising the FNR deficient enterobacterium strain in an inert basesuch as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration can comprise sterile aqueous and non-aqueous injectionsolutions of the FNR deficient enterobacterium strain, whichpreparations are generally isotonic with the blood of the intendedrecipient. These preparations can contain anti-oxidants, buffers,bacteriostats and solutes, which render the formulation isotonic withthe blood of the intended recipient. Aqueous and non-aqueous sterilesuspensions can include suspending agents and thickening agents. Theformulations can be presented in unit\dose or multi-dose containers, forexample sealed ampoules and vials, and can be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or water-for-injection immediatelyprior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets. For example, in one aspect of thepresent invention, there is provided an injectable, stable, sterilecomposition comprising a FNR deficient enterobacterium strain of theinvention, in a unit dosage form in a sealed container. Optionally, thecomposition is provided in the form of a lyophilizate, which is capableof being reconstituted with a suitable pharmaceutically acceptablecarrier to form a liquid composition suitable for injection thereof intoa subject.

Formulations suitable for rectal or vaginal administration can bepresented as suppositories. These can be prepared by admixing the FNRdeficient enterobacterium strain with one or more conventionalexcipients or carriers, for example, cocoa butter, polyethylene glycolor a suppository wax, which are solid at room temperature, but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the FNR deficient FNR deficient enterobacterium strain.

Formulations suitable for topical application to the skin can take theform of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.Carriers that can be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis [see, for example, Pharmaceutical Research 3 (6):318(1986)] and typically take the form of an optionally buffered aqueoussolution. Suitable formulations comprise citrate or bis\tris buffer (pH6) or ethanol/water.

The FNR deficient enterobacterium strain can be formulated for nasaladministration or otherwise administered to the lungs of a subject byany suitable means, for example, by an aerosol suspension of respirableparticles comprising the FNR deficient enterobacterium strain, which thesubject inhales. The respirable particles can be liquid or solid. Theterm “aerosol” includes any gas-borne suspended phase, which is capableof being inhaled into the bronchioles or nasal passages. Specifically,an aerosol includes a gas-borne suspension of droplets, as can beproduced in a metered dose inhaler or nebulizer, or in a mist sprayer.An aerosol also includes a dry powder composition suspended in air orother carrier gas, which can be delivered by insufflation from aninhaler device, for example. See Ganderton & Jones, Drug Delivery to theRespiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviewsin Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al. (1992)J. Pharmacol. Toxicol. Methods 27:143-159. Aerosols of liquid particlescan be produced by any suitable means, such as with a pressure-drivenaerosol nebulizer or an ultrasonic nebulizer, as is known to those ofskill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solidparticles comprising the FNR deficient enterobacterium strain canlikewise be produced with any solid particulate medicament aerosolgenerator, by techniques known in the pharmaceutical art.

In particular embodiments of the invention, administration is bysubcutaneous or intradermal administration. Subcutaneous and intradermaladministration can be by any method known in the art, including but notlimited to injection, gene gun, powderject device, bioject device,microenhancer array, microneedles, and scarification (i.e., abrading thesurface and then applying a solution comprising the FNR deficiententerobacterium strain).

In other embodiments, the FNR deficient enterobacterium strain isadministered intramuscularly, for example, by intramuscular injection.

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Materials and Methods

Bacterial Strains.

Wild-type (WT) serovar Typhimurium (ATCC 14028s) and its isogenic fnrmutant (NC 983) were used throughout the studies described herein. Themutant strain was constructed by transducing the fnr::Tn 10 mutationfrom serovar Typhimurium [SL2986/TN 2958 (fnr::Tn 10)] to strain 14028susing P22 phage (all from the culture collection of S. Libby). Thetransductants were plated on Evans blueuranine agar, and thetetracycline marker was eliminated (Bochner et al., (1980) J. Bacteriol.143:926-933). The Tet^(s) and FNR⁻ phenotypes were confirmed by theinability of NC983 (fnr mutant) to grow on media containing tetracycline(10 μg/ml) and by its inability to grow anaerobically on M9 minimalmedium containing glycerol plus nitrate, respectively. Sequence analysisof fnr and neighboring genes (i.e., ogt and ydaA, respectively) in NC893 showed that the remnant of Tn10 (tnpA) interrupts fnr between bp 106and 107 and has no polar effect on ogt or ydaA (FIG. 1).

For complementation studies, a low-copy-number plasmid expressing fnr(pfnr) was constructed. The complete fnr sequence starting from the stopcodon of ogtA (TGA [indicated in boldface type]) to 21 bp downstream offnr (i.e., a 972 bp fragment) was amplified from WT strain 14028s withthe following primers: fnr-Forward,5′-ATATCCATGGTGAATATACAGGAAAAAGTGC-3′ (an Ncol site is underlined; SEQID NO:1); fnr-Reverse, 5′-ATATATTCAGCTGCATCAATGGTTTAGCTGACG-3′ (a Pvullsite is underlined; SEQ ID NO:2). The PCR product was digested with Ncoland Pvull and ligated into the low-copy-number vector pACYC184 cut withNcol and Pvull. Thus, in the new plasmid (pfnr) the Cmr gene in pACYC184is replaced with the fnr gene. The plasmid (pfnr) was electroporated andmaintained in E. coli DH5α. Transformants were confirmed for Tet^(r) (15μg/ml) and Cms (20 μg/ml) on Luria-Bertani (LB) plates, and the presenceof the fnr gene was confirmed by restriction analysis using EcoRI andHindIII. The plasmid isolated from DH5α was used to complement the fnrmutant. Transformants were selected on LB plates containing tetracycline(15 μg/ml).

Growth Conditions.

The WT and the fnr mutant were grown anaerobically at 37° C. in MOPS(morpholinepropanesulfonic acid)-buffered (100 mM, pH 7.4) LB brothsupplemented with 20 mM D-xylose (LB-MOPS-X). This medium was used inorder to avoid the indirect effects of pH and catabolite repression. ACoy anaerobic chamber (Coy, Ann Arbor, Mich.) and anaerobic gas mixture(10% H2, 5% CO₂, and 85% N₂) were used. All solutions werepreequilibrated for 48 h in the chamber. Cells from frozen stocks wereused to inoculate LB-MOPS-X broth. Cultures were grown for 16 h and usedto inoculate fresh anoxic media. The anaerobic growth kinetics of themutant and the WT strains were similar, and the doubling times of thefnr mutant and the WT were 53.9±1.2 and 45.4±2.9 min, respectively.

RNA Isolation.

Anaerobic cultures were used to inoculate three independent flasks eachcontaining 150 ml of anoxic LB-MOPS-X. The three independent cultureswere grown to an optical density at 600 nm (OD600) of 0.25 to 0.35,pooled, and treated with RNAlater (QIAGEN, Valencia, Calif.) to fix thecells and preserve the quality of the RNA. Total RNA was extracted withthe Rneasy RNA extraction kit (QIAGEN), and the samples were treatedwith RNase-free DNase (Invitrogen, Carlsbad, Calif.). The absence ofcontaminating DNA and the quality of the RNA was confirmed by PCRamplification of known genes and by using agarose gel electrophoresis.Aliquots of the RNA samples were kept at −80° C. for use in themicroarray and quantitative real-time reverse transcription-PCR(qRT-PCR) studies.

Microarray Studies.

Serovar Typhimurium microarray slides were prepared and used aspreviously described in Porwollik, S. et al., “The delta uvrB mutationsin the Ames strains of Salmonella span 15-119 gene” Mutat. Res. 483:1-11(2001). The SuperScript Indirect cDNA labeling system (Invitrogen) wasused to synthesize the cDNA for the hybridizations. Each experimentconsisted of two hybridizations, on two slides, and was carried out inCorning Hybridization Chambers at 42° C. overnight. Dye swapping wasperformed to avoid dye-associated effects on cDNA synthesis. The slideswere washed at increasing stringencies (2×SSC [1×SSC is 0.15 M NaCl plus0.015 M sodium citrate], 0.1% sodium dodecyl sulfate [SDS], 42° C.; 0.1%SSC, 0.1% SDS, room temperature; 0.1% SSC, room temperature). Followinghybridization, the microarrays were scanned for the Cy3 and Cy5fluorescent signals with a ScanArray 4000 microarray scanner from GSILumonics (Watertown, Mass.). The intensity of every spot was codified asthe sum of the intensities of all the pixels within a circle positionedover the spot itself and the background as the sum of the intensities ofan identical number of pixels in the immediate surroundings of thecircled spot.

Data Analysis.

Cy3 and Cy5 values for each spot were normalized over the totalintensity for each dye to account for differences in total intensitybetween the two scanned images. The consistency of the data obtainedfrom the microarray analysis was evaluated by two methods: (i) apair-wise comparison, calculated with a two-tailed Student's t test andanalyzed by the MEAN and TTEST procedures of SAS-STAT statisticalsoftware (SAS Institute, Cary, N.C.) (the effective degrees of freedomfor the t test were calculated as described previously in Satterthwaite,F. E. “An approximate distribution of estimates of variance components”Biometrics Bull. 2:110-114 (1946)); and (ii) a regularized t testfollowed by a posterior probability of differential expression [PPDE(p)] method. These statistical analyses are implemented in the Cyber-Tsoftware package available online at the website of the Institute forGenomics and Bioinformatics of the University of California, Irvine. Thesignal intensity at each spot from the FNR mutant and the WT werebackground subtracted, normalized, and used to calculate the ratio ofgene expression between the two strains. All replicas were combined, andthe median expression ratios and standard deviations were calculated foropen reading frames (ORFs) showing ≧2.5-fold change.

qRT-PCR.

qRT-PCR was used to validate the microarray data, where 19 genes wererandomly chosen from the differentially expressed genes. This techniquewas also used to confirm the expression of a set of selected genes.qRT-PCRs were carried out with the QuantiTect SYBR green RT-PCR kit(QIAGEN) and an iCycler (Bio-Rad, Hercules, Calif.), and the data wereanalyzed by the Bio-Rad Optical System software, version 3.1, accordingto manufacturer specifications. To ensure accurate quantification of themRNA levels, three amplifications for each gene were made with 1:5:25dilutions of the total RNA. Measured mRNA levels were normalized to themRNA levels of the housekeeping gene rpoD (σ70). Normalized values wereused to calculate the ratios of the expression levels in the fnr mutantrelative to the WT.

Logo Graph and Promoter Analysis.

The information matrix for the generation of the FNR logo was producedby using the alignment of the E. coli FNR binding sequences, availableat http://arep.med.harvard.edu/ecoli_matrices/. The alignment of the FNRmotifs from this website did not include the motifs present in the sodAand mutM promoters; therefore, they were included in our analysis. Toaccount for differences in nucleotide usage or slight variations inconsensus sequences, a second alignment was built for serovarTyphimurium using the 5′ regions of the homologous genes originally usedto build the E. coli information matrix. The alignment was used toprepare a new information matrix using the Patser software (version 3d),available at http://rsat.ub.ac.be/rsat/. A graphical representation(FIG. 2) of the matrices through a logo graph was obtained with Weblogosoftware (version 2.8.1, 18 Oct. 2004), available athttp://weblogo.berkeley.edu/.

Motility Assay and Electron Microscopy.

The motilities of the WT, the fnr mutant, and the complementedmutant/pfnr were evaluated under anoxic conditions. Ten microliters ofanaerobically grown (16 h) cells were spotted onto LB-MOPS-X agar (0.6%agar) plates and incubated at 37° C. for 24 h. The diameter of thegrowth halo was used as a measure of motility. Scanning, electronmicroscopy (SEM) was used to examine the morphology of the extracellularsurfaces. WT and fnr cultures were grown anaerobically (OD600, 0.3 to0.4) and centrifuged, and the pellets were resuspended in a fixativesolution (3% glutaraldehyde in 0.1 M phosphate-buffered saline [PBS] [pH7.4]) under anaerobic conditions. The fixed samples were rinsed in 0.1 MPBS buffer, postfixed with 1% osmium tetroxide in 0.1 M PBS for 2 h, andrinsed with PBS, all at 4° C. An aliquot of each sample was filteredthrough a 0.1-μm filter. Each filter was dehydrated through a gradedethanol series (up to 100%), brought to room temperature, critical pointdried with liquid CO₂ (Tousimis Research, Rockville, Md.), placed onstubs, and sputter coated with Au/Pd (Anatech Ltd., Denver, N.C.).Samples were viewed at 15 kV with a JEOL 5900LV SEM (JEOL USA, Peabody,Mass.). Transmission electron microscopy (TEM) and negative stainingwere used to visualize the flagella. WT and fnr cultures were grownanaerobically (OD600, 0.3 to 0.4), and a 20-μl aliquot of each samplewas separately placed on a Formvar-carbon grid. The grids were washedwith 0.1 M sodium acetate (pH 6.6), negatively stained with 2%phosphotungstic acid (PTA), and air dried for 5 min before being viewedat 80 kV with a JEOL JEM-100S TEM (JEOL USA, Peabody, Mass.).

Pathogenicity Assays.

Immunocompetent 6- to 8-week-old C57BL/6 mice and their congenic iNOS−/−and pg91phox^(−/−) immunodeficient mice (bred in the University ofColorado Health Science Center [UCHSC] animal facility according toInstitutional Animal Care and Use Committee guidelines) were used inthis study. Stationary-phase serovar Typhimurium (WT and fnr mutant)cultures grown aerobically in LB-MOPS-X broth were used, and the cellswere diluted in PBS. For oral (p.o.) challenge, groups of 10 mice weregavaged with 5×10⁶ or 5×10⁷ CFU in 200 μl of PBS/mouse. Forintraperitoneal (i.p.) challenge, groups of five mice were inoculatedwith 250 CFU in 500 μl of PBS/mouse. Mortality was scored over a 15- to30-day period.

Macrophage Assay.

Peritoneal macrophages were harvested from C57BL/6 mice andpg91phox^(−/−) immunodeficient mice (bred in the UCHSC animal facility)4 days after intraperitoneal inoculation with 1 mg/ml sodium periodateand used as previously described in DeGroote, M. A. et al. “Periplasmicsuperoxide dismutase protects Salmonella from products of phagocyteNADPH-oxidase and nitric oxide synthase” Proc. Natl. Acad. Sci.94:13997-14001 (1997). Macrophages were challenged (multiplicity ofinfection of 2) for 25 min with the different test strains.Stationary-phase cultures grown aerobically in LB-MOPS-X broth were usedas outlined above. Prior to infection, each strain was opsonized with10% normal mouse serum for 20 min. After the challenge, extracellularbacteria were removed from the monolayers by washing with prewarmed RPMImedium (Cellgro, Herndon, Va.) containing gentamicin (6 mg/ml) (Sigma),the Salmonella-infected macrophages were lysed at indicated time points,and the surviving bacteria were enumerated on LB agar plates. Theresults are expressed as percent survival relative to the number ofviable intracellular bacteria recovered at time zero (i.e., afterwashing and removal of the extracellular bacteria, 25 min afterinfection).

Microarray Data.

The microarray data are accessible via GEO accession number GSE3657 athttp://www.ncbi.nlm.nih.gov/geo (the disclosure of which is incorporatedherein by reference in its entirety).

EXAMPLE 2 Transcriptome Profiling

Out of 4,579 genes, the two-tailed Student t test produced a set of1,664 coding sequences showing significant differences (P<0.05) betweenthe fnr mutant and the WT. The analysis was restricted to include highlyaffected genes (i.e., having a ratio of ≧2.5-fold). Under thisconstraint, 311 genes were differentially expressed in the fnr mutantrelative to the WT; of these, 189 genes were up-regulated and 122 geneswere down-regulated by FNR (Table 3). The 311 FNR-regulated genes wereclassified into clusters of orthologous groups (COGs) as defined athttp://www.ncbi.nlm.nih.gov/COG. Throughout the study levels oftranscription in the fnr mutant were compared to that in the WT strain.Thus, genes repressed by FNR possess values of >1, while genes activatedby FNR have values of <1.

In order to globally validate the microarray data, 19 of the 311differentially expressed genes for qRT-PCR were selected. The measuredlevels of mRNA were normalized to the mRNA levels of the housekeepinggene rpoD. The specific priers used for qRT-PCR and the normalized mRNAlevels are shown in Table 1. The microarray and qRT-PCR data were log₂transformed and plotted (FIG. 3). The correlation between the two setsof data was 0.94 (P<0.05).

To determine whether a binding site for FNR might be present in theregion upstream of the candidate FNR-regulated genes, 5′ regions ofthese genes were searched for the presence of a putative FNR-bindingmotif using a Salmonella logo graph (FIG. 2). One hundred ten out of the189 genes activated by FNR (58%) and 59 out of the 122 genes repressedby FNR (48%) contained at least one putative FNR-binding site.

EXAMPLE 3 FNR as a Repressor

Transcription of the genes required for aerobic metabolism, energygeneration, and nitric oxide detoxification was repressed by FNR. Inparticular, the genes coding for cytochrome c oxidase (cyoABCDE),cytochrome cd complex (cydAB), NADH-dehydrogenase (nuoBCEFJLN),succinyl-coenzyme A (CoA) metabolism (sucBCD), fumarases (fumB, stm0761,and stm0762), and the NO-detoxifying flavohemoglobin (hmpA) wereexpressed at higher levels in the fnr mutant than in the WT (Table 3).Also, genes required for L-lactate metabolism (lld-PRD) and for theproduction of phosphoenolpyruvate (pykF), oxaloacetate (ppc), andacetoacetyl-CoA (yqeF) were expressed at higher levels in the mutantthan in the WT (Table 3).

EXAMPLE 4 FNR as an Activator

Several genes associated with anaerobic metabolism, flagellarbiosynthesis, motility, chemotaxis, and Salmonella pathogenesis wereactivated by FNR. The genes constituting the dms operon, dmsABC(encoding the anaerobic dimethyl sulfoxide reductase), required for theuse of dimethyl sulfoxide (DMSO) as an anaerobic electron acceptor, hadthe lowest expression levels (i.e., −200-, −62-, and −23-fold,respectively) in the fnr mutant relative to the WT (Table 3). Two otheroperons coding for putative anaerobic DMSO reductases (STM4305 toSTM4307 and STM2528 to STM2530) were also under positive control by FNR.The genes required for the conversion of pyruvate to phosphoenolpyruvate(pps), Ac-CoA (aceF), Ac-P (pta), and OAc (ackA), as well as those forthe production of formate (tdcE, yfiD, focA) and D-lactate (IdhA), wereexpressed at lower levels in the fnr mutant than in the WT. In addition,the genes coding for a universal stress protein (ynaF), a ferritinlikeprotein (ftnB), an ATP-dependent helicase (hrpA), and aerotaxis/redoxsensing (aer) were also positively regulated by FNR (Table 3).

The genes for ethanolamine utilization (eut operon) had lower transcriptlevels in the fnr mutant (FIG. 4B). Although the FNR-dependent genes fortetrathionate utilization (ttrABCSR), a major anaerobic electronacceptor, were not affected by the lack of FNR, this was not surprisingsince tetrathionate is also needed to induce expression.

Several of the middle flagellar (class 2) genes (e.g., flgNMDEFGKL andfliZADSTHJLM) and late flagellar (class 3) genes (e.g., cheZYBRMWA,motBA, aer, trg, and tsr) had lower transcript levels in the fnr mutantthan in the WT (FIG. 4C to E). There was no significant difference inthe transcript levels of the early flagellar genes (class 1) flhD andflhC, whose gene products FlhD/FlhC are the master regulators offlagellar biosynthesis (FIG. 4D). In addition, many newly identifiedflagellar genes (i.e., mcpA, mcpC, and cheV) had lower expression levelsin the fnr mutant, while the expression of mcpB was not affected.

Several genes in SPI-1 (e.g., prgKJIH, iagB, sicA, spaPO, invJICBAEGF)had lower levels of expression in the fnr mutant than in the WT (FIG.4A). This region contains genes coding for a type three secretion systemand for proteins required for invasion and interaction with host cells.The data also show that genes belonging to the other SPIs wereunaffected by the lack of FNR. However, the virulence operon srfABC,which is located outside SPI-2 and regulated by a two-componentregulatory system (SsrAB) located on SPI-2 (Waterman et al. (2003) Cell.Microbiol. 5:501-511; Worley et al., (2000) Mol. Microbiol. 36:749-761),was differentially regulated by FNR. The effects of FNR on a subset ofthe above-mentioned invasion and virulence genes were further confirmedby measuring the levels of mRNA in the fnr mutant and the WT strains byqRT-PCR (Table 2).

EXAMPLE 5 Effects of FNR on Motility and Flagella

Expression of the flagellar biosynthesis, motility, and chemotaxis geneswas lower in the fnr mutant than in the WT. Therefore, the WT, fnrmutant was compared to the mutant cells harboring pfnr for motility insoft agar under anaerobic conditions. The data indicate that the fnrmutant was nonmotile and that the lack of motility was complemented(˜75%) by the inclusion of pfnr. The 100% complementation by pfnr isprobably due to extra copies of the global regulator FNR. The WT wasalso compared to the mutant for the presence of flagella by SEM and TEM.Taken together, these data show that the fnr mutant is nonmotile due tothe lack of flagella.

EXAMPLE 6 Effects of FNR on Pathogenicity and Killing by Macrophages

FNR positively regulates the expression of various loci (see Table 3),such as motility and SPI-1 genes that are important determinants forSalmonella pathogenesis, so the virulence of fnr in a murine model ofmucosal and acute infection was tested. In immunocompetent C57BL/6 mice,the fnr mutant was completely attenuated over a 15-day period followingan oral challenge with 5×10⁶ or 5×10⁷ CFU/mouse, while the WT strainkilled all mice within 10 or 12 days, respectively (FIG. 5A). The mutantstrain was also 100% attenuated when 250 CFU/mouse were inoculated i.p.(FIG. 5B). The different Salmonella strains were also tested for theability to survive killing by macrophages (FIG. 6). Similar numbers offnr mutant and WT cells were recovered from the macrophages 25 min afterinfection (designated as time zero postinfection). Data in FIG. 6Aindicate that the lack of FNR resulted in a dramatic reduction in theability of Salmonella to replicate in macrophages. Interestingly, mostof the killing of the WT by macrophages took place during the first 2 hpostinfection (i.e., the WT resisted further killing beyond 2 h), whilethe viability of the fnr mutant continued to decline by 1 log between 2and 20 h postinfection (FIGS. 6A and B). Data in FIG. 6B also show thatthis phenotype is complemented in fnr mutant cells harboring pfnr.Congenic iNOS−/− mice (unable to make NO.) and pg91phox^(−/−) mice(defective in oxidative burst oxidase) were used to examine the roles ofreactive nitrogen and oxygen species (RNS and ROS), respectively, inresistance to an acute systemic infection with FNR-deficient or WTSalmonella. The fnr mutant was as attenuated in iNOS−/− mice as incongenic WT C57BL/6 controls. In sharp contrast, the fnr mutant killedpg91phox^(−/−) mice, albeit at a lower rate than the WT strain (FIGS. 7Aand B). Consistent with the in vivo data, the WT and the isogenic fnrmutant survived to similar extents in NADPH oxidase-deficientmacrophages isolated from pg91phox^(−/−) mice (FIG. 7C).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

TABLE 1 Validation of microarray data using qRT-PCR of randomly selectedgenes relative to the housekeeping gene, rpoD^(a). Ratio of Fragment fnrmutant/WT Log₂ ratio Locus^(b) Name^(c) Primer sequence^(d) SEQ ID NO:(bp)^(e) S. Typhimurium Gene Function^(f) qRT-PCR^(g) Microarray^(h)qRT-PCR^(i) Microarray^(j) STM3217 aer CGTACAACATCTTAATCGTAGC 3 163Aerotaxis sensor receptor; senses 0.190 0.210 −2.4 −2.3TTCGTTCAGATCATTATTACCC 4 cellular redox state or proton motive forceSTM1781 cheM GCCAATTTCAAAAATATGACG 5 114 Methyl-accepting chemotaxisprotein II; 0.036 0.120 −4.8 −3.1 GTCCAGAAACTGAATAAGTTCG 6 aspartatesensor-receptor STM0441 cyoC TATTTAGCTCCATTACCTACGG 7 134 Cytochrome oubiquinol oxidase subunit 153.967 7.096 7.3 2.8 GGAATTCATAGAGTTCCATCC 8III STM1803 dadA TAACCTTTCGCTTTAATACTCC 9 155 D-Amino acid dehydrogenasesubunit 2.835 3.169 1.5 1.7 GATATCAACAATGCCTTTAAGC 10 STM0964 dmsAAGCGTCTTATCAAAGAGTATGG 11 154 Anaerobic dimethyl sulfoxide reductase,0.001 0.005 −9.8 −7.6 TCACCGTAGTGATTAAGATAACC 12 subunit A STM2892 invJTTGCTATCGTCTAAAAATAGGC 13 128 Surface presentation of antigens; 0.2460.182 −2.0 −2.5 TTGATATTATCGTCAGAGATTCC 14 secretory proteins STM2324nuoF GGATATCGAGACACTTGAGC 15 163 NADH dehydrogenase I, chain F 2.8942.600 1.5 1.4 GATTAAATGGGTATTACTGAACG 16 STM0650 STM0650CAACAGCTTATTGATTTAGTGG 17 130 Putative hydrolase, C terminus 0.476 0.219−1.1 −2.2 CTAACGATTTTTCTTCAATGG 18 STM2787 STM2787 AAGCGAATACAGCTATGAACC19 144 Tricarboxylic transport 28.241 6.892 4.8 2.8ATTAGCTTTTGCAGAACATGG 20 STM4463 STM4463 AAGGTATCAGCCAGTCTACG 21 142Putative arginine repressor 0.325 0.181 −1.6 −2.5 CGTATGGATAAGGATAAATTCG22 STM4535 STM4535 TAAGCCAGCAGGTAGATACG 23 139 Putative PTS permease6.053 8.217 2.6 3.0 CGACATAAAGAGATCGATAACC 24 STM2464 eutNAGGACAAATCGTATGTACCG 25 153 Putative detox protein in ethanolamine 0.0620.125 −4.0 −3.0 ACCAGCAGTACCCACTCTCC 26 utilization STM2454 eutRGGTAAAAGAGCAGCATAAAGC 27 118 Putative regulator; ethanolamine operon0.043 0.195 −4.6 −2.4 ATTATCACTCAAGACCTTACGC 28 (AraC/XylS family)STM2470 eutS AATAAAGAACGCATTATTCAGG 29 137 Putative carboxysomestructural protein; 0.049 0.073 −4.3 −3.8 GTTAAAGTCATAATGCCAATCG 30ethanol utilization STM1172 flgM AGCGACATTAATATGGAACG 31 126 Anti-FliA(anti-sigma) factor; also known 0.050 0.174 −4.3 −2.5TTTACTCTGTAAGTAGCTCTGC 32 as RflB protein STM3692 lldPTGATTAAACTCAAGCTGAAAGG 33 189 LctP transporter; L-lactate permease76.492 16.003 6.3 4.0 CCGAAATTTTATAGACAAAGACC 34 STM3693 lldRGAACAGAATATCGTGCAACC 35 153 Putative transcriptional regulator for lct68.378 30.597 6.1 4.9 GAGTCTGATTTTCTCTTTGTCG 36 operon (GntR family)STM1923 motA GGTTATCGGTACAGTTTTCG 37 194 Proton conductor component ofmotor; 0.048 0.092 −4.4 −3.4 TAGATTTTGTGTATTTCGAACG 38 torque generatorSTM4277 nrfA GACTAACTCTCTGTCGAAAACC 39 159 Nitrite reductase;periplasmic 0.051 0.324 −4.3 −1.6 ATTTTATGGTCGGTGTAGAGC 40 cytochromec₅₅₂ ^(a)STM3211 (rpoD) was used as the reference gene where nosignificant change in expression level was observed. The primersequences (5′ to 3′) used for rpoD were as follows: CGATGTCTCTGAAGAAGTGC(forward; SEQ ID NO: 41) and TTCAACCATCTCTTTCTTCG (reverse; SEQ ID NO:42). The size of the fragment generated is 150 bp. ^(b)Location of theopen reading frame (ORF) in the S. Typhimurium LT2 genome.^(c)Respective gene name or symbol. ^(d)For each set, the first primeris the forward primer and the second primer is the reverse primer.^(e)Size of the amplified PCR product. ^(f)Functional classificationaccording to the KEGG (Kyoto Encyclopedia of Genes and Genomes)database. ^(g)Expression levels of quantitative reverse transcriptasepolymerase chain reaction - values shown as the ratio between the fnrmutant and the wild-type; where values <1 indicate that FNR acts as anactivator, and values >1 indicate FNR acts as a repressor.^(h)Expression levels from the microarray data - values shown as theratio between the fnr mutant and the wild-type; where values <1 indicatethat FNR acts as an activator, and values >1 indicate FNR acts as arepressor. ^(i)Expression levels of quantitative reverse transcriptasepolymerase chain reaction comparing the fnr mutant versus thewild-type - shown in signal to log₂ ratio (SLR). ^(j)Expression levelsof microarray data comparing the fnr mutant versus the wild-type - shownin signal to log₂ ratio (SLR).

TABLE 2 qRT-PCR of Selected Invasion and Virulence Genes^(a) SEQ IDFragment Locus^(b) Name^(c) Primer sequence^(d) NO: size (bp)^(e)Ratio^(f) STM2893 invI 5′-CTTCGCTATCAGGATGAGG-3′ 43 161 −9.275′-CGAACAATAGACTGCTTACG-3′ 44 STM2874 prgH 5′-GGCTCGTCAGGTTTTAGC-3′ 45190 −8.45 5′-CTTGCTCATCGTGTTTCG-3′ 46 STM2871 prgK5′-ATTCGCTGGTATCGTCTCC-3′ 47 199 −8.56 5′-GAACCTCGTTCATATACGG-3′ 48STM2886 sicA 5′-GATTACACCATGGGACTGG-3′ 49 207 −3.925′-CAGAGACTCATCTTCAGTACG-3′ 50 STM1593 srfA 5′-AGGCGGCATTTAGTCAGG-3′ 51176 −4.33 5′-GACAGGTAAGCTCCACAGC-3′ 52 STM1594 srfB5′-GGTACCAGAAATACAGATGG-3′ 53 190 −6.55 5′-GCCGATATCAATCGATGC-3′ 54^(a)STM3211 (rpoD) was used as the reference gene where no significantchange in expression level was observed. The primer sequences used forrpoD were as follows: 5′-CGATGTCTCTGAAGAAGTGC-3′ (forward; SEQ ID NO:41) and 5′-TTCAACCATCTCTTTCTTCG-3′ (reverse; SEQ ID NO: 42). The size ofthe fragment generated was 150 bp. ^(b)Location of the open readingframe (ORF) in the S. Typhimurium LT2 genome. ^(c)Respective gene nameor symbol. ^(d)For each set, the first primer is the forward and thesecond primer is the reverse. ^(e)Size of the amplified PCR product.^(f)Ratio of the transcription levels in the fnr mutant relative to thewild-type.

TABLE 3 Differentially expressed genes and the presence/absence ofputative FNR-binding motifs in their 5′ regions. SEQ Cate- STM Gene IDLocus^(a) gory^(b) Name^(c) Function^(d) t value^(e) DF^(f) Prob t^(g)Ratio^(h) Strand^(i) Start^(j) End^(k) Sequence^(l) NO: Score^(m)In(P)^(n) PSLT018 — pefA plasmid-encoded −6.11 5.92 9.16E−04 −2.56fimbriae; major fimbrial subunit PSLT019 — pefB plasmid-encoded −9.866.59 3.46E−05 −4.59 R −337 −316 tttcTTTTTTGATATATGTCTTTTCAtgta 55 5.41−7.58 fimbriae; regulation STM0002 E thrA aspartokinase I, −11.82 7.275.20E−06 −3.33 R −351 −330 GGAATTGGTTGAAAATAAATATatcg 56 5.71 −7.83bifunctional enxyme N-terminal is aspartokinaseI and C-terminal ishomoserine dehydrogenase I STM0041 G STM0041 putative glycosyl −9.215.78 1.15E−04 −3.19 hydrolase STM0042 G STM0042 putative sodium −12.795.16 4.17E−05 −3.32 galactoside symporter STM0153 C aceF pyruvate −7.075.72 4.93E−04 −3.83 D −52 −31 gaatAATGGCTATCGAAATCAAAGTAccgg 57 4.98−7.24 dehydrogenase, dihydro- lipoyltransacetylase component STM0178 GyadI putative PTS −6.35 5.43 1.05E−03 −7.00 D −263 −242tttaTAATATATATTTAATCAATTATtttg 58 5.19 −7.41 enzyme STM0439 H cyoEprotohaeme IX 9.48 5.20 1.77E−04 7.71 D −102 −81tctgGATTATGTGGAACCTCAACTACaaca 59 4.54 −6.9 farnesyltransferase (haeme Obiosynthesis) STM0440 C cyoD cytochrome o 13.11 5.21 3.45E−05 7.05 R−326 −305 tatcGGGATGGAACTCTATGAATTCCatca 60 4.64 −6.98 ubiquinol oxidasesubunit IV STM0441 C cyoC cytochrome o 36.33 5.46 9.96E−08 7.10ubiquinol oxidase subunit III STM0442 C cyoB cytochrome o 22.84 5.648.89E−07 5.05 R −206 −185 aaccTGATTTGTTCAAGGACGTTATTaaca 61 4.18 −6.63ubiquinol oxidase subunit I STM0443 C cyoA cytochrome o 22.81 5.461.25E−06 4.51 D −68 −47 ccgtGGAATTGAGGTCGTTAAATGAGactc 62 5.84 −7.94ubiquinol oxidase subunit II STM0465 S ybaY glycoprotein/ −15.14 5.291.46E−05 −4.75 R −75 −54 ctggTGCATTGATGATAAGGAGAATTgaat 63 5.34 −7.53polysaccharide metabolism STM0467 — ffs signal recognition −9.58 5.211.67E−04 −4.45 particle, RNA component STM0650 G STM0650 putativehydrolase −9.87 5.46 1.10E−04 −4.56 D −55 −34aaggGAAAATGATTATGAGCAATGAGactt 64 6.95 −8.95 C-terminus STM0659 O hscCputative heatshock −15.49 8.16 2.46E−07 −2.82 R −128 −107gcgtGACATTGATAAAGATCACCACGccag 65 5.25 −7.46 protein, homolog of hsp70in Hsc66 subfamily STM0662 E gltL ABC superfamily 12.96 5.46 2.61E−054.39 R −309 −288 actgGCAGTCGATGAAGCTCATTATTctgc 66 5.97 −8.06(atp_bind), glutamate/aspartate transporter STM0663 E gltK ABCsuperfamily 12.08 8.19 1.67E−06 2.81 (membrane), glutamate/aspartatetransporter STM0664 E gltJ ABC superfamily 12.47 7.16 4.09E−06 2.67 D−62 −41 tttcGGAGTAGATGTATGTCAATAGActgg 67 4.58 −6.93 (membrane),glutamate/aspartate transporter STM0665 E gltI ABC superfamily 10.655.77 5.20E−05 4.24 D −216 −195 taggATTTTTGCCTCTGAACGGTGCGgcgc 68 5.67−7.8 (bind_prot), glutamate/aspartate transporter STM0699 R STM0699putative −15.11 6.35 3.25E−06 −4.22 R −30 −9ggaaGTCACTGATATAGCAGAAATACtggc 69 6.99 −8.99 cytoplasmic protein STM0728L nei endonuclease VIII 16.01 6.47 1.90E−06 2.57 D −80 −59tccaTTAATCAACTGTTAACAAAGGAtatt 70 5.12 −7.35 removing oxidizedpyrimidines may also remove oxidized purines in absence of MutY and Fpg[EC: 3.2.—.—] STM0737 C sucB 2-oxoglutarate 14.92 9.44 7.01E−08 2.75dehydrogenase (dihydro- lipoyltranssuccinase E2 component) STM0738 CsucC succinyl-CoA 21.50 5.79 9.65E−07 4.03 D −39 −18acatGAATATCAGGCAAAACAACTTTttgc 71 6.69 −8.71 synthetase, beta subunitSTM0739 C sucD succinyl-CoA 23.03 5.62 8.87E−07 4.66 synthetase, alphasubunit STM0740 C cydA cytochrome d 17.22 6.09 2.13E−06 2.55 R −307 −286gccaTAAATTGATCGCTGTCGAAAAAagca 72 10.38 −13.11 terminal oxidase,polypeptide subunit I [EC: 1.10.3.—] STM0741 C cydB cytochrome d 21.165.67 1.30E−06 3.74 D −169 −148 cagaATTGTTCCTGATGTTCAAATTTgcac 73 5.62−7.76 terminal oxidase polypeptide subunit II STM0742 S ybgT putativeouter 11.38 7.56 5.01E−06 4.34 R −165 −144tgttCGTACCGATCATTCTGATCTACacca 74 4.83 −7.12 membrane lipoproteinSTM0743 S ybgE putative inner 13.92 9.36 1.44E−07 3.48 R −102 −81cacgTGCACTGTTGGAAGAGGTTATCcgac 75 4.31 −6.72 membrane lipoproteinSTM0759 — ybgS putative homeobox −14.35 5.04 2.80E−05 −4.60 proteinSTM0761 C STM0761 fumarate hydratase 6.55 5.53 8.38E−04 4.48 Class Ianaerobic STM0762 C STM0762 fumarate 8.26 5.15 3.67E−04 5.69 D −340 −319agttTATTTTTCTTTCTATCAAATAAtgtc 76 6.33 −8.37 hydratase, alpha subunitSTM0781 P modA ABC superfamily −22.42 7.25 5.78E−08 −3.12 R −140 −119tttaATCGTTAATGGGTATGAATAACcgct 77 6.43 −8.47 (peri_perm), molybdatetransporter STM0790 — hutU pseudogene; 9.49 5.45 1.36E−04 5.44frameshift relative to Pseudomonas putida urocanate hydratase (HUTU)(SW: P25080) STM0791 E hutH histidine ammonialyase 19.06 6.84 3.51E−074.55 STM0828 E glnQ ABC superfamily 8.23 6.06 1.66E−04 2.86 (atp_bind),glutamine high- affinity transporter STM0830 E glnH ABC superfamily 9.605.47 1.26E−04 3.70 (bind_prot), glutamine high- affinity transporterSTM0853 — yliH putative −15.57 6.53 2.08E−06 −3.33 cytoplasmic proteinSTM0907 R aSTM0907 Fels-1 prophage; −6.72 5.39 8.16E−04 −3.12 putativechitinase STM0912 O aSTM0912 Fels-1 prophage; 11.94 7.50 3.81E−06 3.40protease subunits of ATP-dependent proteases, ClpP family STM0964 C dmsAanaerobic dimethyl −18.73 5.00 7.95E−06 −200.85 D −151 −130ctacTTTTTCGATATATATCAGACTTtata 78 7.44 −9.43 sulfoxide reductase,subunit A STM0965 C dmsB anaerobic dimethyl −53.46 5.29 1.93E−08 −62.55sulfoxide reductase, subunit B STM0966 R dmsC anaerobic dimethyl −28.935.04 8.52E−07 −23.60 R −260 −239 gtaaGAAACCGATTTGCGTCGAATCCtgcc 79 8.79−10.93 sulfoxide reductase, subunit C STM0972 — STM0972 homologous to5.62 6.48 1.04E−03 3.07 D −256 −235 aataATTCTCAACATAATTCAGATGTgtcc 804.68 −7.01 secreted protein sopD STM0974 P focA putative FNT −7.49 5.773.53E−04 −3.19 R −129 −108 ggcgAGATATGATCTATATCAAATTCtcat 81 8.35 −10.41family, formate transporter (formate channel 1) STM0989 — STM0989 mukFprotein −10.99 5.11 9.47E−05 −4.90 R −279 −258cgtgCGCGCTGAAATGCGTTAACATGatcc 82 4 −6.5 (killing factor KicB) STM1118 —yccJ putative −9.37 5.50 1.38E−04 −4.94 cytoplasmic protein STM1119 RwraB trp-repressor −10.54 5.13 1.14E−04 −6.45 D −167 −146attaATTATTGTTATAAATCAAAGAAatgg 83 9.3 −11.57 binding protein STM1123 SSTM1123 putative 4.28 6.28 4.69E−03 3.13 periplasmic protein STM1124 —putA bifunctional in 24.50 6.20 2.07E−07 7.57 plasma membrane prolinedehydrogenase and pyrroline-5- carboxylate dehydrogenase OR in cytoplasma transcriptional repressor STM1125 E putP SSS family, major 14.50 5.461.44E−05 7.16 D −195 −174 tgtaAATGGTGTGTTAAATCGATTGTgaat 84 7.78 −9.79sodium/proline symporter STM1126 — phoH PhoB-dependent, 9.41 6.883.56E−05 2.63 NA NA NA NA NA NA ATP-binding pho regulon componentSTM1128 E STM1128 putative −18.20 6.39 9.58E−07 −4.78 R −289 −268cctgAAGCCTGTTTGAACGCAATATCggat 85 5.25 −7.45 sodium/glucosecotransporter STM1129 G STM1129 putative inner −15.72 5.52 8.59E−06−6.50 membrane protein STM1130 S STM1130 putative inner −9.85 5.151.56E−04 −11.85 membrane protein STM1131 — STM1131 putative outer −10.505.84 5.25E−05 −4.67 D −58 −37 cggaGTATTTTATGAAAATCAACAAAtatc 86 7.06−9.06 membrane protein STM1132 G STM1132 putative sugar −9.42 5.291.67E−04 −6.20 R −120 −99 ttcgGCAATTGATATGACTTAAAAATtaat 87 10.03 −12.57transort protein STM1133 R STM1133 putative −14.98 5.29 1.56E−05 −5.56 D−90 −69 cgtgAAAGTTTTCAATCAACAAAAGAattt 88 4.6 −6.94 dehydrogenases andrelated proteins STM1138 — ycdZ putative inner −9.09 5.03 2.62E−04−11.00 D −106 −85 agaaTAATGTGATGTAAATCACCCTTaact 89 5.45 −7.62 membraneprotein STM1171 N flgN flagellar −12.69 5.24 3.93E−05 −7.85biosynthesis: belived to be export chaperone for FlgK and FlgL STM1172 KflgM anti-FliA (anti- −13.69 5.30 2.46E−05 −5.75 R −72 −51cgtaACCCTCGATGAGGATAAATAAAtgag 90 5.44 −7.61 sigma) factor; also knownas RflB protein STM1176 N flgD flagellar −11.76 5.49 4.21E−05 −2.94biosynthesis, initiation of hook assembly STM1177 N flgE flagellar−14.54 5.88 7.83E−06 −3.56 biosynthesis, hook protein STM1178 N flgFflagellar −7.59 6.06 2.58E−04 −2.81 biosynthesis, cell- proximal portionof basal-body rod STM1179 N flgG flagellar −7.52 5.56 4.10E−04 −3.50biosynthesis, cell- distal portion of basal-body rod STM1183 N flgKflagellar −11.80 5.20 5.95E−05 −5.67 biosynthesis, hook- filamentjunction protein 1 STM1184 N flgL flagellar −11.04 5.33 7.16E−05 −4.18biosynthesis; hook- filament junction protein STM1227 E pepT putativepeptidase −13.20 6.24 8.57E−06 −2.67 T(aminotripeptidase) STM1254 —STM1254 putative outer −8.06 5.65 2.64E−04 −3.82 membrane lipoproteinSTM1271 P yeaR putative 13.89 5.66 1.37E−05 4.25 R −284 −263gcaaTTCTTTGATTGGCCTTCTTTTCgtcg 91 4.81 −7.1 cytoplasmic protein STM1272— yoaG putative 6.92 7.98 1.23E−04 2.87 D −225 −204cggaAGAGATCATGGTGATCAATGCCggcg 92 8.55 −10.65 cytoplasmic proteinSTM1300 — STM1300 putative −8.78 5.08 2.93E−04 −4.83 D −256 −235ggttGTATTTGCGTTTTATCAGAATAtgta 93 6.36 −8.4 periplasmic protein STM1301L STM1301 putative mutator −9.26 5.65 1.27E−04 −3.36 MutT proteinSTM1349 G pps phosphoenolpyruvate −19.01 5.69 2.28E−06 −3.45 D −58 −37aggaTTGTTCGATGTCCAACAATGGCtcgt 94 5.74 −7.86 synthase STM1378 G pykFpyruvate kinase I 14.46 5.51 1.36E−05 2.55 (formerly F), fructosestimulated STM1489 H ynfK putative −11.20 5.23 7.52E−05 −8.40 R −140−119 aactCAAGCTGATTGCCCTTGCCATAtctt 95 4.73 −7.04 dethiobiotin synthaseSTM1498 C STM1498 putative dimethyl −21.41 5.10 3.44E−06 −27.55 R −177−156 atacAAATCTGGTGGAAATCGAAAAAatct 96 4.87 −7.15 sulphoxide reductaseSTM1499 C STM1499 putative dimethyl −19.08 5.35 3.98E−06 −8.27 D −101−80 ataaTTTCGTTATAGTTATCAATATAtagc 97 4.38 −6.78 sulphoxide reductase,chain A1 STM1509 — ydfZ putative −14.41 5.58 1.25E−05 −7.62 R −161 −140ccgtGAGCTTGATCAAAAACAAAAAAaatt 98 8.84 −10.98 cytoplasmic proteinSTM1538 C STM1538 putative 11.55 5.78 3.28E−05 3.96 hydrogenase-1 largesubunit STM1539 C STM1539 putative 8.66 5.44 2.21E−04 3.53 hydrogenase-1small subunit STM1562 — STM1562 putative −11.30 5.28 6.68E−05 −4.99 D−217 −196 tgctTTATTCATCAAACATCAAAATCagtc 99 6.71 −8.73 periplasmictransport protein STM1564 — yddX putative −10.75 8.27 3.83E−06 −2.93cytoplasmic protein STM1568 C fdnI formate −28.97 6.32 5.81E−08 −3.98 R−119 −98 tcgcCGGTCTGATTTACCACTACATCggta 100 7.14 −9.14 dehydrogenase-N,cytochrome B556(Fdn) gamma subunit, nitrate- inducible STM1569 C fdnHformate −9.33 8.93 6.68E−06 −2.50 dehydrogenase, iron-sulfur subunit(formate dehydrogenase beta subunit) [EC: 1.2.1.2] STM1593 — srfA ssrABactivated −5.82 6.28 9.63E−04 −2.52 D −185 −164acccTGATTTAACTTACGTCAAGTGGaaac 101 5.8 −7.91 gene STM1594 S srfB ssrABactivated −14.70 6.14 5.14E−06 −2.88 D −58 −37tgccTGATTTTATGTTGGTCAATCTGtgtg 102 5.31 −7.5 gene STM1626 N trgmethyl-accepting −13.30 5.46 2.28E−05 −5.72 chemotaxis protein III,ribose and galactose sensor receptor STM1640 S ydcF putative inner−11.44 5.63 4.15E−05 −5.72 membrane protein STM1641 L hrpA helicase,ATP- −21.19 6.25 4.68E−07 −20.97 R −217 −196gtgcCCCGTTGCTTGTTGACACTTTAttca 103 4.72 −7.04 dependent STM1642 I acpDacyl carrier protein −11.32 7.81 4.05E−06 −4.01 D −107 −86tgaaTAAAGTGTCAACAAGCAACGGGgcac 104 4.72 −7.04 phosphodiesterase STM1647C ldhA fermentative D- −16.29 5.95 3.65E−06 −24.42 D −139 −118tcatTATATGTATGACTATCAATTATtttt 105 5.05 −7.29 lactate dehydrogenase,NAD-dependent STM1648 O hslJ heat shock protein −8.34 5.41 2.75E−04−5.00 R −74 −53 ttaaCTATCAGATTACAGAGAATATCaatg 106 4.11 −6.57 hslJSTM1650 — STM1650 putative reverse −5.38 7.56 7.97E−04 −3.52transcriptase STM1651 C nifJ putative pyruvate- −8.67 6.43 8.87E−05−4.97 flavodoxin oxidoreductase STM1652 T ynaF putative universal −20.675.01 4.81E−06 −116.15 R −282 −261 ttatTGAATTAAACGGTAACATCTCTtttt 107 4.7−7.02 stress protein STM1653 P STM1653 putative membrane −10.25 5.855.92E−05 −6.56 transporter of cations STM1657 N STM1657 putative methyl-−9.41 6.29 6.15E−05 −4.01 D −52 −31 caaaAATGTTGAGAAATATCAGCGTCagga 1088.58 −10.68 accepting chemotaxis protein STM1658 S ydaL putative Smr−28.29 6.75 2.87E−08 −4.01 domain STM1659 L ogt O-6-alkylguanine- −6.796.25 4.20E−04 −2.92 DNA/cysteine- protein methyltransferase STM1660 —fnr transcriptional −8.24 5.04 4.11E−04 −6.55 D −88 −67tgttAAAATTGACAAATATCAATTACggct 109 11.43 −14.93 regulation of aerobic,anaerobic respiration, osmotic balance (CRP family) STM1688 K pspC phageshock 11.96 6.86 7.63E−06 3.23 R −45 −24 gggtGGAATCAATCTGAATAAAAAACtatg110 6.11 −8.18 protein; regulatory gene, activates expression of pspoperon with PspB STM1706 J yciH putative translation 9.58 5.71 9.91E−054.32 initiation factor SUI1 STM1732 M ompW outer membrane −6.72 5.071.05E−03 −8.36 R −172 −151 gttcTAAATTAATCTGGATCAATAAAtgtt 111 8.33−10.39 protein W; colicin S4 receptor; putative transporter STM1746 —oppA ABC superfamily 16.43 6.25 2.23E−06 3.83 R −290 −269atttCACATTGTTGATAAGTATTTTCattt 112 5.36 −7.54 (periplasm), oligopeptidetransport protein with chaperone properties STM1767 T narL responseregulator 11.78 6.53 1.22E−05 2.78 in two-component regulatory systemwith NarX (or NarQ), regulates anaerobic respiration and fermentation(LuxR/UhpA familiy) STM1781 P ychM putative SuIP −10.77 6.07 3.49E−05−3.33 R −230 −209 acgaAGAATCGATTTCCGCCATGTTCgagc 113 5.29 −7.49 familytransport protein STM1795 E STM1795 putative homologue 12.42 5.463.28E−05 5.33 R −302 −281 acatAACATTGATACATGTCGTTATCataa 114 6.57 −8.6of glutamic dehyrogenase STM1798 — ycgR putative inner −14.17 5.948.40E−06 −4.09 D −267 −246 tggcGATAACGCCGGCAATCAAACCAaaaa 115 6.66 −8.68membrane protein STM1803 E dadA D-amino acid 10.10 8.96 3.40E−06 3.16 R−261 −240 cctcCACATTGAACGGCAAAAAATCGggta 116 4.58 −6.92 dehydrogenasesubunit STM1831 G manY Sugar Specific PTS 15.20 5.98 5.28E−06 3.73 R−146 −125 atccGAAACTGAAAATGATGGATTTAattg 117 4.99 −7.24 family, mannose-specific enzyme IIC STM1832 G manZ Sugar Specific PTS 14.15 9.241.42E−07 2.96 D −277 −256 ttggTTATGCGATGGTTATCAATATGatgc 118 7.21 −9.2family, mannose- specific enzyme IID STM1915 N cheZ chemotactic −9.595.76 9.42E−05 −3.56 response; CheY protein phophatase STM1916 T cheYchemotaxis −9.32 5.69 1.18E−04 −3.74 D −111 −90agcaGATGTTGGCGAAAATCAGTGCCggac 119 8.6 −10.7 regulator, transmitschemoreceptor signals to flagellar motor components STM1917 N cheBmethyl esterase, −8.37 5.74 2.00E−04 −4.33 response regulator forchemotaxis (cheA sensor) STM1918 N cheR glutamate −22.41 8.99 3.40E−09−3.32 methyltransferase, response regulator for chemotaxis STM1919 NcheM methyl accepting −18.86 5.13 6.12E−06 −8.31 chemotaxis protein II,aspartate sensor-receptor STM1920 N cheW purine-binding −14.43 5.321.82E−05 −4.31 D −296 −275 gggcATTGTTGTGATCCTGCAAAGCGcggg 120 4.39 −6.78chemotaxis protein; regulation STM1921 N cheA sensory histitine −16.276.02 3.32E−06 −4.67 D −39 −18 ggatATTAGCGATTTTTATCAGACATtttt 121 4.84−7.12 protein kinase, transduces signal between chemo- signal receptorsand CheB and CheY STM1922 N motB enables flagellar −15.03 5.33 1.43E−05−7.00 R −177 −156 atgcGCCGCCGATTGCCGTGGAATTTggtc 122 5.72 −7.84 motorrotation, linking torque machinery to cell wall STM1923 N motA protonconductor −5.97 5.07 1.80E−03 −10.82 component of motor, torquegenerator STM1932 P ftnB ferritin-like protein −21.51 5.01 3.92E−06−21.11 D −95 −74 tctcGTCTGTCATGCACATCAACACTttct 123 4.24 −6.67 STM1955 —fliZ putative regulator −15.76 5.35 1.09E−05 −6.25 D −331 −310acagGAAAACCCGTTACATCAACTGCtgga 124 5.78 −7.9 of FliA STM1956 K fliAsigma F (sigma 28) −21.86 8.81 5.60E−09 −5.96 R −64 −43acgcAGGGCTGTTTATCGTGAATTCActgt 125 4.87 −7.15 factor of RNA polymerase,transcription of late flagellar genes (class 3a and 3b operons) STM1960N fliD flagellar −15.69 6.81 1.35E−06 −4.45 R −82 −61cttaACTACTGTTTGCAATCAAAAAGgaag 126 4.63 −6.97 biosynthesis; filamentcapping protein; enables filament assembly STM1961 O fliS flagellar−9.80 5.27 1.40E−04 −5.10 R −231 −210 acgcCACGCTGAAAAGCCTGACAAAAcagt 1274.08 −6.56 biosynthesis; repressor of class 3a and 3b operons (RflAactivity) STM1962 — fliT flagellar −7.66 5.16 5.25E−04 −6.03biosynthesis; possible export chaperone for FliD STM1971 N fliHflagellar −8.61 6.32 1.01E−04 −2.94 biosynthesis; possible export offlagellar proteins STM1973 N fliJ flagellar fliJ protein −7.71 6.361.88E−04 −4.27 STM1975 N fliL flagellar −8.94 5.55 1.68E−04 −2.79biosynthesis STM1976 N fliM flagellar −9.19 5.26 1.95E−04 −3.07 R −161−140 aacaAAAACTGATTGCCGCCATTAAAgaga 128 5.62 −7.76 biosynthesis,component of motor switch and energizing STM1978 N fliO flagellar −5.216.02 1.98E−03 −2.76 biosynthesis STM2059 S yeeX putative 8.96 6.904.79E−05 2.75 cytoplasmic protein STM2183 F cdd cytidine/deoxycytidine−17.85 5.47 4.66E−06 −7.71 R −157 −136 atttTTCATTGAAGTTTCACAAGTTGcata129 5.31 −7.51 deaminase STM2186 E STM2186 putative NADPH- −15.60 5.647.43E−06 −4.18 D −269 −248 cttcTTTTTTATCGTTAATCTATTTAttat 130 5.46 −7.63dependent glutamate synthase beta chain or related oxidoreductaseSTM2187 F yeiA putative −15.69 5.44 9.74E−06 −4.05 dihydropyrimidinedehydrogenase STM2277 F nrdA ribonucleoside 26.74 5.26 8.07E−07 11.15 D−60 −39 ggtaGAAAACCACATGAATCAGAGTCtgct 131 4.2 −6.64 diphosphatereductase 1, alpha subunit STM2278 F nrdB ribonucleoside- 29.51 5.691.92E−07 9.57 D −68 −47 tcccATAAAGGATTCACTTCAATGGCatac 132 6.02 −8.11diphosphate reductase 1, beta subunit STM2279 C yfaE putative ferredoxin6.33 5.30 1.17E−03 3.37 R −297 −276 acggTTCGATGATCGGCCTGAATAAAgata 1335.02 −7.27 STM2280 G STM2280 putative permease 11.04 6.42 2.06E−05 4.32STM2287 — STM2287 putative 4.59 6.33 3.24E−03 3.46 R −279 −258ggggATAACTGAATATCCCCAATAATaatt 134 4.46 −6.84 cytoplasmic proteinSTM2314 T STM2314 putative −25.76 5.48 6.21E−07 −6.99 chemotaxis signaltransduction protein STM2315 R yfbK putative von −15.60 9.01 7.88E−08−2.84 Willebrand factor, vWF type A domain STM2316 — nuoN NADH 8.29 6.131.50E−04 2.67 R −334 −313 tggtGCCGGTGATTACCGTGATCTCCacct 135 4.26 −6.69dehydrogenase I chain N STM2318 C nuoL NADH 8.02 7.14 8.05E−05 2.54 R−261 −240 tgttTATGCTGATTGGGCTGGAAATCatga 136 5.57 −7.71 dehydrogenase Ichain L STM2320 C nuoJ NADH 11.27 6.55 1.58E−05 2.54 dehydrogenase Ichain J [EC: 1.6.5.3] STM2324 C nuoF NADH 9.85 7.93 1.01E−05 2.60dehydrogenase I chain F STM2325 C nuoE NADH 8.07 8.32 3.28E−05 2.65dehydrogenase I chain E STM2326 C nuoC NADH 22.56 6.50 2.04E−07 3.34dehydrogenase I chain C, D STM2327 C nuoB NADH 11.71 6.20 1.86E−05 2.52dehydrogenase I chain B [EC: 1.6.5.3] STM2334 R yfbT putative 18.20 6.341.03E−06 3.49 R −76 −55 gagcGCGAATGAAATCAATCAAATCAttaa 137 5.55 −7.7phosphatase STM2335 S yfbU putative 6.74 5.61 6.87E−04 3.39 cytoplasmicprotein STM2337 C ackA acetate kinase A −16.97 6.34 1.60E−06 −3.51 R−147 −126 tcctGCGCATGATGTTAATCATAAATgtca 138 4.76 −7.07 (propionatekinase 2) STM2338 C pta phosphotransacetylase −6.43 5.94 6.96E−04 −3.17STM2340 G STM2340 putative 11.33 5.19 7.42E−05 6.60 D −82 −61gctcAATGAGGCCATTCATCAACTGGaggt 139 4.18 −6.62 transketolase STM2341 GSTM2341 putative 23.38 5.42 1.19E−06 6.47 transketolase STM2342 SSTM2342 putative inner 9.98 5.52 9.77E−05 5.22 R −276 −255aaaaAGTATTAAAGAAACTCAATATTgacg 140 6.82 −8.83 membrane protein STM2343 GSTM2343 putative 10.35 6.09 4.30E−05 3.34 D −64 −43tttaAAAGGTGACAATAATGAAAATCatgg 141 4.64 −6.97 cytoplasmic proteinSTM2409 F nupC NUP family, −15.30 5.45 1.09E−05 −3.91 D −347 −326gtttATTGATAATGATTATCAAGTGCattt 142 5.87 −7.97 nucleoside transportSTM2454 K eutR putative regulator −12.08 5.35 4.34E−05 −5.13ethanolamine operon (AraC/XylS family) STM2455 Q eutK putative −9.275.21 1.97E−04 −6.77 D −61 −40 aacgGAGGCTGCCAATGATCAATGCCctgg 143 4.45−6.83 carboxysome structural protein, ethanolamine utilization STM2456 EeutL putative −9.22 5.22 1.99E−04 −6.68 carboxysome structural protein,ethanolamine utilization STM2457 E eutC ethanolamine −16.21 5.566.85E−06 −7.07 ammonia-lyase, light chain STM2458 E eutB ethanolamine−17.30 5.54 4.94E−06 −6.33 D −315 −294 ccgcATTACTCACGGTCATCAACGCGctga144 5.38 −7.56 ammonia-lyase, heavy chain STM2459 E eutA CPPZ-55 −19.265.16 5.24E−06 −6.17 prophage; chaperonin in ethanolamine utilizationSTM2460 E eutH putative transport −17.75 5.32 6.09E−06 −6.29 protein,ethanolamine utilization STM2462 E eutJ paral putative −7.74 5.055.52E−04 −7.30 heatshock protein (Hsp70) STM2463 C eutE putativealdehyde −18.52 5.34 4.73E−06 −7.02 R −64 −43aaatAGGATTGAACATCATGAATCAAcagg 145 4.88 −7.15 oxidoreductase inethanolamine utilization STM2464 Q eutN putative detox −14.11 5.142.65E−05 −8.00 D −194 −173 tggaAGAAGTGTTCCCGATCAGCTTCaaag 146 5.21 −7.42protein in ethanolamine utilization STM2465 Q eutM putative detox −28.265.11 8.21E−07 −11.02 R −250 −229 ctatCGCGCTGTTGGGCCGCTAATTCaggg 147 4.58−6.93 protein in ethanolamine utilization STM2466 C eutD putative −15.835.11 1.53E−05 −10.36 phosphotransacetylase in ethanolamine utilizationSTM2467 E eutT putative cobalamin −16.88 5.92 3.13E−06 −6.59 R −237 −216cgtgGACGCTGAACTACGACGAAATCgaca 148 6.89 −8.9 adenosyltransferase,ethanolamine utilization STM2468 E eutQ putative −18.58 5.26 5.33E−06−7.60 ethanolamine utilization protein STM2469 E eutP putative −20.065.13 4.47E−06 −9.38 R −247 −226 aaacGGCGATGATCGCTGGCGATTTAgcga 149 4.71−7.03 ethanolamine utilization protein STM2470 E eutS putative −10.435.05 1.32E−04 −13.70 R −154 −133 ttctCTTAGTGATCTACCTCACCTTTtaca 150 5.95−8.05 carboxysome structural protein, ethanol utilization STM2479 E aegAputative −7.82 7.39 7.90E−05 −3.37 R −187 −166gaaaTAAATTGATCTGCCACAGGTTCtgga 151 7.26 −9.26 oxidoreductase STM2530 CSTM2530 putative anaerobic −10.14 7.00 1.96E−05 −3.67 D −158 −137ttatGAATTTCATTTAATTTAAAGTTaatg 152 6.79 −8.8 dimethylsulfoxide reductaseSTM2556 C hmpA dihydropteridine 15.99 5.95 4.09E−06 3.01 R −95 −74agatGCATTTGATATACATCATTAGAtttt 153 6.24 −8.3 reductase 2 and nitricoxide dioxygenase activity STM2558 E cadB APC family, −6.65 5.191.01E−03 −6.43 D −219 −198 tatgTTAATTCAAAAAAATCAATCTAtcag 154 6.78 −8.8lysine/cadaverine transport protein STM2559 E cadA lysine −10.67 5.181.02E−04 −5.58 R −221 −200 tcgtCAGTCTGATCATCCTGATGTTCtacg 155 5.74 −7.86decarboxylase 1 STM2646 R yfiD putative formate −26.43 5.69 3.54E−07−5.08 D −179 −158 ggttTTTATTGATTTAAATCAAAGAAtgaa 156 10.76 −13.71acetyltransferase STM2733 — STM2733 Fels-2 prophage: −5.44 8.92 4.26E−04−2.72 similar to E. coli retron Ec67 STM2786 S STM2786 tricarboxylic19.84 5.84 1.38E−06 8.21 transport STM2787 — STM2787 tricarboxylic 10.549.96 1.01E−06 6.89 transport STM2788 S STM2788 tricarboxylic 11.85 5.464.19E−05 3.46 R −107 −86 ttgaCCGGCTGCTTGATGTCACCTTAcctc 157 4.26 −6.68transport STM2795 S ygaU putative LysM −3.59 5.59 1.31E−02 −2.98 D −55−34 aggtGAATATGGGACTTTTCAATTTTgtaa 158 5.43 −7.6 domain STM2851 C hycChydrogenase 3, −5.41 8.55 5.07E−04 −3.49 membrane subunit (part of FHLcomplex) STM2855 O hypB hydrogenase-3 −14.31 6.06 6.74E−06 −3.12 R −143−122 cataGAGATTGATGAAACTGAAGATTaatg 159 9.23 −11.47 accessory protein,assembly of metallocenter STM2856 O hypC putative −9.09 6.18 8.40E−05−2.56 hydrogenase expression/formation protein STM2857 O hypD putative−8.01 5.29 3.76E−04 −2.97 hydrogenase expression/formation proteinSTM2871 U prgK cell invasion −15.27 7.06 1.15E−06 −3.25 protein;lipoprotein, may link inner and outer membranes STM2872 — prgJ cellinvasion −9.60 5.35 1.43E−04 −4.65 R −344 −323agccCACTTTAATTTAACGTAAATAAggaa 160 5.69 −7.82 protein; cytoplasmicSTM2873 — prgI cell invasion −12.42 5.80 2.13E−05 −4.16 R −83 −62agccCACTTTAATTTAACGTAAATAAggaa 161 5.69 −7.82 protein; cytoplasmicSTM2874 — prgH cell invasion −16.99 6.31 1.65E−06 −3.85 protein STM2877M iagB cell invasion −4.52 5.49 5.01E−03 −3.55 R −86 −65ccgcTTGATTAAATTACGGTAAAATCtgag 162 4.68 −7 protein STM2886 R sicAsurface −10.24 5.19 1.24E−04 −2.80 D −57 −36ggagTAAGTAATGGATTATCAAAATAatgt 163 4.34 −6.75 presentation of antigens;secretory proteins STM2890 U spaP surface −5.39 5.53 2.17E−03 −4.27 D−88 −67 cttaGGCGTTGAGATCCATGAATGGCtgag 164 4.61 −6.95 presentation ofantigens; secretory proteins STM2891 N spaO surface −8.43 5.86 1.72E−04−3.67 presentation of antigens; secretory proteins STM2892 — invJsurface −7.06 5.22 7.34E−04 −5.51 D −239 −218tttaGAACTCCAGATTATACAAATTCagga 165 4.22 −6.66 presentation of antigens;secretory proteins STM2893 — invI surface −12.02 5.46 3.91E−05 −4.44 R−190 −169 gcttTTCATTGACTTGGGAGAATATCgtcc 166 6.09 −8.16 presentation ofantigens; secretory proteins STM2894 N invC surface −6.62 6.04 5.54E−04−2.79 presentation of antigens; secretory proteins STM2895 — invBsurface −10.44 5.50 7.85E−05 −3.88 R −267 −246ccgcTAATTTGATGGATCTCATTACActta 167 8.41 −10.48 presentation of antigens;secretory proteins STM2896 U invA invasion protein −6.84 5.81 5.50E−04−3.34 STM2897 — invE invasion protein −5.52 6.12 1.40E−03 −3.09 R −29 −8tccgGTATTTCATTTTCCAGAATATTgtcc 168 4.35 −6.76 STM2898 N invG invasionprotein; −6.82 5.89 5.30E−04 −3.48 D −126 −105cacaTTTTTCTAGTGAGATCAAAGAGctga 169 7.49 −9.49 outer membrane STM2899 KinvF invasion protein −5.45 5.63 1.95E−03 −3.57 STM2983 — ygdI putativelipoprotein −7.97 6.00 2.09E−04 −3.64 STM3019 I yqeF putative acetyl-CoA10.03 6.84 2.46E−05 2.58 R −249 −228 ctatTGATTTGCTGTGGAACAAGAAAacgc 1704.54 −6.9 acetyltransferase STM3131 S STM3131 putative −17.74 9.671.07E−08 −7.24 R −77 −56 actgCCACCTGATCAACAAGGAGATAaatc 171 5.09 −7.32cytoplasmic protein STM3136 G STM3136 putative D- 9.94 5.61 8.96E−052.88 mannonate oxidoreductase STM3138 N STM3138 putative methyl- −8.985.43 1.84E−04 −4.76 accepting chemotaxis protein STM3155 — STM3155putative −7.23 7.42 1.31E−04 −2.81 R −78 −57gtatACCACTGATCGTAAAGGATATTtagt 172 7.28 −9.28 cytoplasmic proteinSTM3216 N STM3216 putative methyl- −15.35 7.19 9.30E−07 −3.37 R −280−259 tctaCCTATTAATAGGTATAAACTCAgtta 173 5.59 −7.73 accepting chemotaxisprotein STM3217 T aer aerotaxis sensor −12.23 5.32 4.29E−05 −4.77 D −238−217 aaagGTTGTCCACGCTAAACAATTTCataa 174 8.02 −10.04 receptor, sensescellular redox state or proton motive force STM3225 E ygjU putative17.69 9.71 1.04E−08 3.68 dicarboxylate permease STM3238 S yhaN putativeinner −6.92 6.10 4.20E−04 −3.18 R −146 −125aaggCGCTTCGTTGTACCTGATTATTatta 175 4.55 −6.9 membrane protein STM3240 EtdcG L-serine −16.72 5.57 5.68E−06 −4.80 R −249 −228ggatGCGATTGAACATCCGGAAAACTaccc 176 6.02 −8.11 deaminase STM3241 C tdcEpyruvate formate- −9.34 10.00 2.95E−06 −2.66 lyase 4/2- ketobutyrateformate-lyase STM3242 C tdcD propionate −14.83 7.09 1.35E−06 −4.10 R−147 −126 tgacCATCCTGAATATTGTCTACAAAttgt 177 4.53 −6.89 kinase/acetatekinase II, anaerobic STM3245 K tdcA transcriptional −17.03 5.41 6.59E−06−6.04 D −239 −218 cctgTTTTTTGATTGAAATCAGGCTAagtt 178 8.48 −10.57activator of tdc operon (LysR family) STM3248 I garR tartronate 15.785.93 4.53E−06 4.16 semialdehyde reductase (TSAR) STM3273 I yhbT putativelipid carrier −9.33 5.49 1.43E−04 −2.71 D −237 −216acgcAAAATTGTTAACGAACAGGGATttta 179 5.52 −7.68 protein STM3274 O yhbUputative protease −10.06 5.51 9.38E−05 −10.68 R −103 −82taaaATCCCTGTTCGTTAACAATTTTgcgt 180 5.52 −7.68 STM3275 — yhbV putativeprotease −13.35 5.95 1.16E−05 −10.88 STM3334 F STM3334 putative cytosine−8.46 6.65 8.47E−05 −2.58 D −277 −256 agtgATTATTGCCGACTATCTGTTGAaccg 1814 −6.5 deaminase STM3338 G nanT MFS family, sialic −19.92 5.67 1.83E−06−3.02 acid transport protein STM3545 R yhhX putative 14.13 5.91 8.84E−063.21 oxidoreductase STM3547 — STM3547 putative 54.21 5.60 7.77E−09 6.06transcriptional regulator of sugar metabolism STM3548 — STM3548 putative40.36 5.24 9.82E−08 9.29 R −327 −306 gcttGCTTATGATGGCACACAATTCAtcta 1824.98 −7.24 cytoplasmic protein STM3549 S STM3549 putative inner 13.965.28 2.25E−05 7.22 membrane protein STM3550 R STM3550 putative 22.455.17 2.34E−06 7.40 phosphotriesterase STM3576 P zntA P-type ATPase 9.905.54 9.92E−05 2.55 R −261 −240 cgacGCTGCTGTTCATCGGCAATATCgtct 183 5.02−7.27 family, Pb/Cd/Zn/Hg transporting ATPase [EC: 3.6.3.3 3.6.3.5]STM3577 N tcp methyl-accepting −22.22 5.71 9.23E−07 −5.64 R −126 −105agcgTGATTTGATGTAAGGTTAATTTttat 184 6.53 −8.56 transmembranecitrate/phenol chemoreceptor STM3598 E STM3598 putative L- −8.27 6.471.13E−04 −3.70 asparaginase STM3599 R STM3599 putative inner −7.19 5.117.38E−04 −9.14 D −108 −87 cacaATAGGTTACGTCCCTCAATGTAaagc 185 4.29 −6.71membrane protein STM3600 G STM3600 putative sugar −9.76 5.07 1.77E−04−12.63 R −322 −301 aacgCGCGTTAAACTTCCTGAAAAAAtatg 186 5 −7.25 kinases,ribokinase family STM3601 M STM3601 putative −26.75 5.09 1.12E−06 −11.94R −266 −245 cgcaACAGTTGATTGTCGGCGATAAAatac 187 7.59 −9.59 phosphosugarisomerase STM3611 T yhjH putative −16.54 5.11 1.23E−05 −7.89 Diguanylatecyclase/phosphodiesterase domain 3 STM3626 E dppF ABC superfamily 9.035.90 1.14E−04 5.86 (atp_bind), dipeptide transport protein STM3627 EdppD ABC superfamily 8.88 5.24 2.36E−04 7.65 D −45 −24ggcgTTATTAAATGTAGATCAATTATcggt 188 8.18 −10.23 (atp_bind), dipeptidetransport protein STM3628 E dppC ABC superfamily 16.86 5.71 4.35E−064.09 (membrane), dipeptide transport protein 2 STM3629 E dppB ABCsuperfamily 11.76 5.16 6.34E−05 12.45 D −55 −34ttcgGGTTATGTTGCAGTTCATTCTCcgac 189 4.22 −6.66 (membrane), dipeptidetransport protein 1 STM3630 E dppA ABC superfamily 9.06 5.06 2.56E−049.90 (peri_perm), dipeptide transport protein STM3690 — STM3690 putativeinner −15.85 9.97 2.15E−08 −5.67 R −305 −284ccgcAAAATTAAATAACATTATCATCcctg 190 4.96 −7.22 membrane lipoproteinSTM3692 C lldP LctP transporter, L- 9.84 5.02 1.81E−04 16.00 D −160 −139tgtcATTATCCATACACAACAATATTggca 191 6.37 −8.41 lactate permease STM3693 KlldR putative 12.30 5.04 5.98E−05 30.60 transcriptional regulator forlct operon (GntR family) STM3694 C lldD L-lactate 27.93 5.02 1.05E−0628.33 dehydrogenase STM3695 J yibK putative 9.64 9.95 2.31E−06 2.95tRNA/rRNA methyltransferase STM3708 E tdh threonine 3- 7.11 6.532.66E−04 3.16 dehydrogenase STM3709 H kbl 2-amino-3- 12.63 6.75 6.02E−062.86 D −298 −277 taacGATATTGCTGCCGATAAAGCCCgcgc 192 5.12 −7.34ketobutyrate CoA ligase (glycine acetyltransferase) STM3750 G yicJputative GPH −13.67 7.06 2.44E−06 −2.67 family transport protein STM3801G dsdX putative Gnt family 14.70 7.68 6.70E−07 3.42 R −22 −1gcacGCTGCTGATCAGCATCGTGTT 193 5.63 −7.77 transport protein STM3802 EdsdA D-serine 17.71 5.05 9.69E−06 7.90 deaminase (dehydratase) STM3808 —ibpB small heat shock 6.72 5.53 7.44E−04 2.85 protein STM3820 P STM3820putative −12.03 6.07 1.83E−05 −5.87 D −165 −144tgtaATTATTGATACCAATCAATATCcatg 194 11 −14.14 cytochrome c peroxidaseSTM3831 R yidA putative hydrolase 19.09 7.62 1.03E−07 4.38 D −220 −199acggTATTTTCTGTTTGATTAATGAGgtta 195 7.22 −9.21 of the HAD superfamilySTM3861 M glmS L-glutamine:D- 8.04 6.11 1.80E−04 2.89 R −24 −3cgcgCAGCGTGATGTAGCTGAAATCCt 196 4.19 −6.63 fructose-6- phosphateaminotransferase STM3862 M glmU N-acetyl 7.97 5.70 2.67E−04 2.78glucosamine-1- phosphate uridyltransferase and glucosamine- 1-phosphateacetyl transferase STM3909 E ilvC ketol-acid 10.62 5.70 5.68E−05 3.38reductoisomerase STM4004 H hemN O2-independent 10.06 5.66 8.07E−05 3.82coproporphyrinogen III oxidase STM4007 E glnA glutamine 19.35 5.323.91E−06 5.97 synthetase STM4034 O fdhE putative formate 9.93 6.015.99E−05 3.12 dehydrogenase formation protein ? Mn_fn STM4035 C fdoIformate 8.84 5.80 1.40E−04 3.60 dehydrogenase, cytochrome B556 (FDO)subunit STM4036 C fdoH formate 21.65 8.93 5.02E−09 2.85 dehydrogenase-O,Fe—S subunit STM4037 C fdoG formate 7.75 5.52 3.62E−04 2.73dehydrogenase STM4062 G pfkA 6- 12.62 9.16 4.23E−07 2.97 R −181 −160cattTGGCCTGACCTGAATCAATTCAgcag 197 5.19 −7.4 phosphofructokinase ISTM4078 G yneB putative fructose- 15.91 7.71 3.57E−07 2.57 R −51 −30aagaATGGCTGATTTAGATGATATTAaaga 198 5.53 −7.68 1,6-bisphosphate aldolaseSTM4085 G glpX unknown function 6.62 5.50 8.16E−04 3.20 R −60 −39ctacGAGTTTGTTATGAGACGAGAACttgc 199 5.56 −7.71 in glycerol metabolismSTM4109 G talC putative 19.34 8.13 4.38E−08 4.72 D −221 −200tcatTATGCTGACGCTTAACAAACACgccg 200 4.79 −7.09 transaldolase STM4110 GptsA General PTS 7.39 6.01 3.14E−04 2.66 D −257 −236tactGGATTTTTGTAATATCAGTATAaaaa 201 5.49 −7.65 family, enzyme I STM4113 GfrwB PTS system 5.65 5.64 1.62E−03 2.89 D −83 −62tttaGATTTTGAGATGAATTAAGCGAggaa 202 4.79 −7.09 fructose-like IIBcomponent 1 STM4119 C ppc phosphoenolpyruvate 9.54 5.26 1.62E−04 3.40carboxylase STM4126 C udhA soluble pyridine 13.35 6.46 6.04E−06 3.17nucleotide transhydrogenase STM4229 G malE ABC superfamily 56.71 9.343.52E−13 7.00 (bind_prot) maltose transport protein, substraterecognition for transport and chemotaxis STM4231 G lamB phage lambda26.26 5.36 7.19E−07 11.38 receptor protein; maltose high- affinityreceptor, facilitates diffusion of maltose and maltoseoligosaccharidesSTM4240 S yjbJ putative −6.01 5.16 1.64E−03 −4.24 cytoplasmic proteinSTM4277 P nrfA nitrite reductase −7.36 5.12 6.58E−04 −3.09 R −198 −177acttACAATTGATTAAAGACAACATTttaa 203 11.55 −15.16 periplasmic cytochromec(552) STM4278 — nrfB formate-dependent −7.18 7.31 1.46E−04 −3.69 D −262−241 gtttGAATATGCAACAAATCAACGCGgaga 204 6.12 −8.19 nitrite reductase; apenta-haeme cytochrome c STM4298 G melA alpha- 25.01 5.44 7.96E−07 7.21galactosidase STM4299 G melB GPH family, 24.57 5.24 1.29E−06 6.66 D −327−306 cgatGATGCAGACCAACATCAACGTGcaaa 205 4.94 −7.21 melibiose permease IISTM4300 C fumB fumarase B 9.38 5.99 8.37E−05 2.69 R −216 −195tgccGGGTTTGATTGGCGTGAGCGTCtcct 206 4.17 −6.62 (fumarase hydratase classI), anaerobic isozyme STM4301 R dcuB Dcu family, 12.46 5.83 2.01E−053.38 R −155 −134 ctggCGCATTGAATATTCGCCATTTCctga 207 5.55 −7.7 anaerobicC4- dicarboxylate transporter STM4305 — STM4305 putative anaerobic−15.43 5.16 1.62E−05 −10.14 dimethyl sulfoxide reductase, subunit ASTM4306 C STM4306 putative anaerobic −7.45 5.19 5.86E−04 −7.59 R −65 −44cttaAGGAGTGATGTACGATGAAACAgtat 208 4.33 −6.73 dimethyl sulfoxidereductase, subunit B STM4307 R STM4307 putative anaerobic −13.21 9.881.33E−07 −2.90 dimethyl sulfoxide reductase, subunit C STM4308 R STM4308putative component −7.84 6.73 1.27E−04 −2.55 of anaerobic dehydrogenasesSTM4398 E cycA APC family, D- 13.44 6.79 3.81E−06 2.54 D −151 −130gccgATTCTTACCTAATATCGATGAGtcct 209 5.02 −7.27 alanine/D- serine/glycinetransport protein STM4399 D ytfE putative cell 7.64 6.16 2.31E−04 2.96morphogenesis STM4439 C cybC cytochrome b(562) 23.97 7.61 1.92E−08 4.26R −317 −296 attcTGGGTTGAAAATGGTGAAATCCagta 210 5.06 −7.3 STM4452 F nrdDanaerobic −12.74 6.51 7.62E−06 −3.17 R −304 −283ttttTACCTTGTTCTACATCAATAAAattg 211 7.97 −9.99 ribonucleoside-triphosphate reductase STM4462 — yjgG putative −5.83 5.39 1.64E−03 −3.63cytoplasmic protein STM4463 E STM4463 putative arginine −8.53 5.332.64E−04 −5.52 repressor STM4469 E argI ornithine −12.54 9.04 5.08E−07−3.36 carbamoyltransferase 1 STM4510 M STM4510 putative aspartate −6.505.16 1.14E−03 −6.35 D −115 −94 gcatTTTTTATATACACATCAAGTTGatag 212 6.58−8.61 racemase STM4511 K yjiE putative −8.28 5.18 3.54E−04 −6.79transcriptional regulator, LysR family STM4512 — iadA isoaspartyl −16.845.28 8.66E−06 −6.59 R −75 −54 gcagCTTATTGTTTAATAAGGAGTTAtcat 213 4.52−6.88 dipeptidase STM4513 S yjiG putative permease −12.81 5.10 4.53E−05−8.61 STM4514 — yjiH putative inner −31.77 6.17 4.45E−08 −6.56 R −338−317 gcgtGAAATTGACTAACGTCAAATTTattt 214 9.1 −11.31 membrane proteinSTM4526 V hsdR endonuclease R, −10.25 5.85 5.93E−05 −3.28 R −153 −132attgTTCGTTGATCACACACAATATGaagt 215 5.93 −8.02 host restriction STM4533 Ntsr methyl-accepting −4.07 6.18 6.19E−03 −2.75 D −301 −280cgcgTAAAGTTAGGTAAATCAGTGAGtggt 216 7.31 −9.31 chemotaxis protein I,serine sensor receptor STM4535 G STM4535 putative PTS 18.00 6.011.87E−06 8.22 D −179 −158 agaaCTTATCGAGCAAGATCAACAGTttta 217 4.23 −6.66permease STM4536 G STM4536 putative PTS 15.55 5.53 8.94E−06 4.50permease STM4537 G STM4537 putative PTS 29.73 6.56 3.04E−08 6.57 R −172−151 gttaGCGGATGAAATGACTCAACTTCggga 218 5.44 −7.61 permease STM4538 GSTM4538 putative PTS 12.87 5.17 4.03E−05 7.25 permease STM4539 M STM4539putative 7.10 5.07 8.07E−04 8.20 R −277 −256cggcCTCCATGATTGATATCACCATTccca 219 5.1 −7.33 glucosamine- fructose-6-phosphate aminotransferase STM4540 — STM4540 putative 10.51 5.269.95E−05 4.89 glucosamine- fructose-6- phosphate aminotransferaseSTM4561 R osmY hyperosmotically −11.35 5.80 3.54E−05 −5.19 D −163 −142tcacGAATGTGATGCCAGTCATTGACttca 220 4.12 −6.59 inducible periplasmicprotein, RpoS-dependent stationary phase gene STM4565 O yjjW pyruvateformate −15.96 9.55 3.30E−08 −3.71 D −34 −13gcgcTTTAGTCAGTAAGATCATTGCGtttt 221 5.28 −7.48 lyase activating enzymeSTM4566 — yjjI putative −20.43 5.09 4.43E−06 −17.28 R −181 −160actgTAATGAGATCTGAATCAAATTAtccc 222 4.68 −7 cytoplasmic protein STM4567 FdeoC 2-deoxyribose-5- −6.83 6.15 4.34E−04 −2.91 D −184 −163gggaTAATTTGATTCAGATCTCATTAcagt 223 4.68 −7 phosphate aldolase STM4568 FdeoA thymidine −8.83 6.49 7.51E−05 −2.76 phosphorylase ^(a)Location ofthe open reading frame (ORF) in the S. Typhimurium LT2 genome.^(b)Functional category assigned to the gene by the National Center forBiotechnology Information, Cluster of Orthologous Genes (COGs). Thedesignations of functional categories are as follows: C, energyproduction and conversion, D, cell cycle control and mitosis, E, aminoacid metabolism and transport, F, nucleotide metabolism and transport,G, carbohydrate metabolism and transport, H, coenzyme metabolism andtransport, I, lipid metabolism and transport, J, translation, K,transcription, L, replication, recombination, and repair, M, cellwall/membrane/envelope biogenesis, N, Cell motility, O,post-translational modification, protein turnover, chaperone functions,P, inorganic ion transport and metabolism, Q, secondary metabolitesbiosynthesis, transport, and catabolism, R, general functionalprediction only (typically, prediction of biochemical activity), S,function unknown, T, signal transduction mechanisms, U, intracellulartrafficking and secretion, V, defense mechanisms, —, not in COGs.^(c)Respective gene name or symbol. ^(d)Functional classificationaccording to the KEGG (Kyoto Encyclopedia of Genes and Genomes)database. ^(e)The numerical value of t for the t test (statisticalmethod). ^(f)The degrees of freedom employed for the analysis of eachgene. ^(g)The probability associated with the t test for each gene.^(h)Ratio between the expression level of the fnr mutant relative to thewild-type. ^(i)The strand on which the motif has been localized. R,reverse; D, direct; NA, not available in the Regulatory SequenceAnalysis Tools (RSAT) database (the locus identity was not recognized),and a blank cell indicates that no motif was present. ^(j)The startingposition of the putative motif. The positions are relative to the regionsearched and span from −300 to +50 relative to the starting ATG. ^(k)Theending position of the putative motif. The positions are relative to theregion searched and span from −300 to +50 relative to the starting ATG.^(l)The sequence of the HIGHEST RANKING putative motif (capitalizedletters) and 4 base pairs (bps) flanking either side of the region(lower case letters). A blank cell indicates that no motif was present.All of the sequences are reported from the 5′ to the 3′ end of the ORF(Open Reading Frame) analyzed. ^(m)The score indicating the similarityof the motif to the information matrix. The cutoff used was a scorehigher than 4.00 or a In(P) lower than −6.5. ^(n)The natural logarithmof the probability that the putative motif is randomly similar to theinformation matrix. The cutoff used was a score higher than 4.00 or aIn(P) lower than −6.5.

We claim:
 1. A pharmaceutical composition comprising an attenuatedSalmonella comprising an attenuating deletion mutation in theFumarate-Nitrate Reductase (fnr) gene and an adjuvant in apharmaceutically acceptable carrier, wherein the attenuating deletionmutation in the fnr gene results in an attenuated Salmonella that (i)lacks flagella and is non-motile, and (ii) has reduced expression ofSPI-1 genes, thereby resulting in reduced virulence.
 2. Thepharmaceutical composition of claim 1, wherein the Salmonella isSalmonella enterica serovar Typhimurium (S. Typhimurium).
 3. A method ofinducing an immune response in a subject comprising administering to thesubject the pharmaceutical composition of claim 1.